KR20060080227A - Pharmaceutical formulations, methods and dosing regimens for the treatment and prevention of acute coronary syndromes - Google Patents

Abstract

The invention provides methods and formulations for treating and preventing acute coronary syndromes. The methods of the instant invention provide safe and effective doses of an Apolipoprotein A-I Milano: phospholipid complex to reduce and stabilize atherosclerotic plaque. Pharmaceutical fomrulations of the Apo A-I Milano: phospholipid complexes are also provided.

Description

PHARMACEUTICAL FORMULATIONS, METHODS AND DOSING REGIMENS FOR THE TREATMENT AND PREVENTION OF ACUTE CORONARY SYNDROMES}

The present invention relates to novel agents and methods for the treatment or prevention of acute coronary syndromes. In a preferred embodiment, the formulation is in a single unit dosage form having the specified pH, osmolality and purity. In a preferred method, the formulation is administered once a week, once a month or once a year, in a dosage range of about 1 mg / kg to about 100 mg / kg per dose. In addition, doses and dosing regimens and the specific diseases for which such doses or dosage regimens are to be used are described. The formulation and method uses Apolipoprotein A-I Milano: phospholipid complex.

The management and treatment of myocardial infarction has been an emphasis on techniques including hemodynamic monitoring and balloon catheters since the first half of the twentieth century, as well as an increased focus on thrombolytic therapy. It has been changed rapidly (Antman and Braunwald, "Acute Myocardial Infarction" in Heart Disease, A Textbook of Cardiovascular Medicine, 6 ^th edition, vol. 2, Braunwald et al ., eds, 2001, WB Saunders Company, Philadelphia). Therapeutic approaches to treating cardiovascular disease have entailed a greater understanding of primary lesions and have been tremendously developed over the last 100 years.

Nearly all myocardial infarctions usually result from coronary atherosclerosis with overlapping coronary thrombosis. Slowly accumulating plaques can be asymptomatic due to the development of side vessels. However, atherosclerotic plaques, particularly those rich in lipids, are susceptible to sudden plaque destruction. Plaque destruction and associated endothelial damage result in the release of mediators such as thromboxane A ₂ , serotonin, adenosine diphosphate, thrombin, platelet activating factor, tissue factor and oxygen-derived free radicals. These mediators promote platelet aggregation and mechanical occlusion, leading to thrombus formation that inhibits blood flow and oxygen supply. Persistent and severe inhibition of myocardial oxygen supply can lead to acute myocardial infarction (see Rioufol et. al ., 2002, Circulation 106: 804, Timmis, 2003, Heart 89: 1268-72).

Primary atherosclerosis pharmacotherapy was first a chronic treatment that prevented or slowed the development of atherosclerosis plaques by focusing on lowering LDL or "bad cholesterol" as a therapeutic endpoint. Statin therapies, for example, greatly benefit the improved cardiovascular health, but side effects such as rhabdomyolysis remain an obstacle. In addition, statins have little effect in reducing acute and unstable atherosclerosis plaques in, for example, an ischemic episode process in acute situations. Acute treatment is mostly based on surgical interventions such as thrombolytics (eg tPA) and percutaneous coronary angioplasty (PTCA) and coronary artery bypass graft (CABG). Thrombolytics provide relief by reducing or eliminating obstructive thrombus, but they do not change the original lesion. Interventions like PTCA carry their own risks and are often unsuitable for patients in acute conditions. Thus, current pharmacological treatments provide little benefit to patients once unstable plaques are present as a risk factor (Newton and Krause 2002, Atherosclerosis S3: 31-38).

HDL therapy is known as a new therapeutic regime for dyslipidemia and atherosclerosis. Apolipoprotein AI Milan as described above (Apo AI Milan) is due to the paradoxical fact that carriers, which are variants of this apolipoprotein, have low HDL ("good cholesterol") levels and a reduced risk of cardiovascular disease. It is of interest (see Franceschini et al ., 1980, J. Clin . Invest. 66: 892-900, Weisgraber et al ., 1983, J. Biol . Chem . 258: 2508-2513, Franceschini et al ., 1985, Atherosclerosis 58: 159-174, Franceschini et al ., 1987, Arteriosclerosis 7: 426-435). Apo AI Milano homodimer has been shown to reduce endovascular thickening in cholesterol-fed rabbits (Ameli et al ., 1994, Circulation , 90: 1935-41; And Soma et al ., 1995, Cir . Res . 76: 405-11). In ApoE deficient mice, atherosclerosis lesions were reduced by both several low doses and a single high dose Apo AI Milan: Lipid complex (Shah et. al ., 1998, Circulation 97: 780-85; And Shah et al ., 2001, Circulation 103: 3047-50). Plaque degeneration in rabbits was also shown by one local infusion of the Apo AI Milano: phospholipid complex at doses of 500 mg and 1000 mg of protein per rabbit (Chiesa et. al ., 2002, Cir . Res . 90: 974-80). Lesions induced in rabbit models are mostly macrophages and do not represent more complex lesions in humans. Thus, it is not clear whether similar therapies will be effective against complex plaques in human atherosclerosis (Li et al. al ., 1999, Arterioscler Thromb Vasc Biol 19: 378-383; And Shah et al ., 2001, Circulation 103: 3047-50).

Despite an improved understanding of the pathophysiology of myocardial infarction and advances in HDL therapy, safe and effective dose, dosing regimens for the prophylactic and therapeutic use of Apo AI Milano or Apo AI Milano: phospholipide complexes in humans And pharmaceutical formulations are still needed.

Summary of the Invention

The present invention provides for the treatment and prevention of cardiovascular diseases or related diseases including atherosclerosis, acute coronary syndrome, ischemia, ischemic reperfusion injury, angina and myocardial infarction, and reduction or stabilization of atherosclerosis plaques, occlusion Methods and pharmaceutical formulations for plaque reduction and accelerated cholesterol outflow in vascularized blood vessels are provided. Accordingly, the present invention provides for the treatment and prevention of cardiovascular diseases or related diseases including atherosclerosis, acute coronary syndrome, ischemia, ischemic reperfusion injury, angina and myocardial infarction, and reduction or stabilization of atherosclerosis plaques, occlusion Dosage and dosage regimens for use of the Apo AI Milano: Phosporipid complex and Plasma Regimen and Apo AI Milano: Phospolipid complex for reducing plaque in the blood vessels and promoting cholesterol outflow. The methods and formulations described herein provide for the rapid reduction of unstable atherosclerosis plaques that, when left untreated or treated by conventional methods, can lead to ischemic seizures including acute coronary syndrome by destruction or Causes stabilization.

Applicants prefer that exogenously produced HDL mimetic, such as Apo AI Milano as the Apo AI Milano: phospholipid complex, can be used for atherosclerosis, acute coronary syndrome, ischemia, ischemic reperfusion injury, angina and myocardium. It has been determined to provide a unique approach for the treatment and prevention of cardiovascular or related diseases including, but not limited to, infarction, or for the reduction or stabilization of atherosclerotic plaques in narrowed or occluded blood vessels. This method, including the dose and dosage regimens of the present invention and pharmaceutical formulations, provides a safe and effective non-surgical treatment that rapidly promotes cholesterol outflow and migration from atherosclerosis plaques. Promoting rapid cholesterol outflow decreases the volume of atherosclerosis in one or more diseased vessels. Decreased atherosclerosis (a mass of degenerative thickening of the endometrial plaques present in atherosclerotic vessels) causes blood flow to be larger and reduces the risk of ischemia, including unstable anxiety, myocardial infarction and acute coronary syndromes.

Conventional atherosclerosis therapy prior to the present invention providing a pharmaceutical formulation of Apo A-IM and a prescribed dose for the treatment and prevention of acute coronary syndrome, atherosclerosis, angina, myocardial infarction and ischemic reperfusion injury Has focused on lowering LDL or "bad cholesterol" as a therapeutic endpoint. These conventional LDL therapies, such as treatment with statins, may require several months of treatment before the LDL serum levels are lowered in the subject. Common treatments for acute or emergency cardiovascular disease are mostly based on surgical interventions such as percutaneous coronary angioplasty (PTCA) and coronary artery bypass graft (CABG). Surgical intervention, however, is often contraindicated in some patients in emergencies, especially those with poor general health. Moreover, these conventional methods do not provide effective non-surgical pharmacological treatment in acute situations. In addition, conventional therapies have focused on treating symptoms of cardiovascular disease and acute coronary syndromes, especially in the circulatory system. Conventional therapies, as described above and described in more detail below, do not treat or prevent the underlying causes of cardiovascular disease and acute coronary syndrome, stabilization, reduction and modification of plaque in the vessel wall, reduce cholesterol, The focus was on reducing formation and reducing blood pressure.

Pharmaceutical formulations and methods, including the dosages and dosing schedules provided herein, are safe and effective and act quickly to reduce or stabilize atherosclerosis plaques. The doses described herein are rapid acting, providing a reduction in atherosclerosis plaques in a short period of time by several weeks by promoting cholesterol transport. In certain embodiments, the doses described herein can be used to treat a subject suffering from acute coronary syndrome or a disease associated with ischemia or vascular occlusion. In certain embodiments, the doses described herein can be used to prevent disease progression once atherosclerosis plaques present a risk in a subject. The progression of the prevented disease may be further to prevent the destruction of unstable atherosclerosis plaques, which may be an occlusion of blood vessels or may lead to ischemic conditions, including acute coronary syndrome and ischemic reperfusion injury.

The methods and pharmaceutical formulations provided herein are so effective that a single dose of 45 mg / kg can promote the migration of cholesterol from atherosclerotic plaques. In certain embodiments, a single high dose, eg, about 45 mg / kg, of the Apo A-I Milano: Phosporipid complex or a pharmaceutical formulation thereof may be administered to a subject. In certain embodiments, one or more high doses of Apo AI Milano: phospholipid complex or a pharmaceutical formulation thereof is administered to a subject, followed by one or more identical doses (45 mg / kg) or low dose, eg, About 1 mg / kg, about 3 mg / kg, about 5 mg / kg, about 10 mg / kg or about 15 mg / kg may be administered. For example, the subject may be administered two doses of 45 mg / kg, followed by an additional dose of 10 mg / kg. In addition, an opposite regime may be used, in which a low dose is administered followed by a high dose. Of course, in the case of an acute disease state higher doses are preferably used first. In certain embodiments, the subject is treated once a week with the Apo AI Milano: Phospholipide complex or pharmaceutical formulation thereof for several weeks, and then maintains open non-occluded blood vessels and risks of plaque destruction and vascular side effects. It may be treated in an intermittent manner, for example, twice a year, three times a year, once a year, or every two years as needed to reduce the risk.

In certain embodiments, the method for the treatment or prevention of acute coronary syndrome comprises administering the Apo AI Milano: phospholipide complex or a pharmaceutical formulation thereof approximately daily, approximately every other day, approximately every three days, approximately every four days, approximately five Administration to a subject in need thereof every day, approximately every 6 days, approximately every 7 days, approximately every 8 to 10 days, or approximately every 11 to 14 days. In a preferred embodiment, the Apo A-I Milano: phospholipide complex or pharmaceutical formulation thereof can be administered approximately every seven days. In certain embodiments, administration of the Apo A-I Milano: Phospholipide complex or pharmaceutical formulation thereof may be a single administration. In certain embodiments, the administration is for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7-12 weeks, about 13-24 weeks, or about 25-52 weeks Can be continued. In certain preferred embodiments, administration of the Apo A-I Milano: phospholipide complex or pharmaceutical formulation thereof takes place approximately every seven days for about five weeks. In certain embodiments, administration can be intermittent, for example, after approximately 5 weeks. For example, the subject may be treated once a week for about 5 weeks, then about 3 to about 4 times over the next year. In certain embodiments, the pharmaceutical formulations described herein may be administered to a subject intermittently to maintain the openness of blood vessels. For example, a dose of about 15 mg / kg may be administered as a pharmaceutical formulation for about 7 weeks, approximately every 10 days, and then treated, for example, after about 26 weeks, or after about 52 weeks. Other embodiments, including dosing schedules and the use of methods in combination with conventional drug therapies and surgical interventions, are described below.

The present invention provides a pharmaceutical formulation for administering apolipoprotein AI Milan (Apo AI Milan) for the treatment or prevention of acute coronary syndrome and ischemic reperfusion injury. In a preferred embodiment, Apo AI Milan forms a complex with lipids for the methods described herein. For example, lipids are phospholipids, preferably 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (also referred to as 1-palmitoyl-2-oleoyl-phosphatidylcholine or POPC). May be referred to). In the most preferred embodiment, the Apo AI Milano: POPC complex can be a pharmaceutical formulation. In another preferred embodiment, the Apo AI Milano: phospholipide complex or pharmaceutical formulation thereof can be administered to a subject in need of a dose of about 1 mg to about 100 mg of protein per kg.

In certain embodiments, the method may comprise administering a dose of Apo A-I Milano: Phosporipid complex or an injectable or liquid pharmaceutical formulation thereof for the treatment of acute coronary syndrome. In a preferred embodiment, the Apo A-IM: phospholipid complex or pharmaceutical formulation thereof can be administered to a subject in need of a dose of about 1 mg (protein) / kg to about 100 mg (protein) / kg. In certain embodiments, the method may comprise preventing or treating acute coronary syndrome by administering the Apo A-I Milano: Phospholipide complex or a pharmaceutical formulation thereof by intravenous infusion. In certain embodiments, the method may be administered by intravenous push infusion. Intravenous rapid infusion means intravenous administration of Apo A-I Milano: phospholipid or a pharmaceutical formulation thereof within 5 minutes, over a short period of time such as, for example, 2-5 minutes. In certain embodiments, administration of the Apo A-I Milano: phospholipid complex or pharmaceutical formulation thereof may be by continuous intravenous infusion. Continuous intravenous infusion means that the Apo A-I Milano: phospholipid complex or pharmaceutical formulation thereof is administered continuously over a period of time longer than 2-5 minutes, eg, from about 30 minutes to about 3 hours. Continuous intravenous infusion can be administered with the aid of an infusion pump or device. In certain embodiments, administration of Apo AI Milano: phospholipid or a pharmaceutical formulation thereof comprises continuous intravenous infusion and intravenous rapid infusion of the Apo AI Milano: phospholipide complex or pharmaceutical formulation thereof (“bolus dose” ) "). The bolus dose can be administered before, after or during continuous infusion. In certain embodiments, administration of Apo A-I Milano or Lipid Complex or pharmaceutical formulations thereof may be in combination with other drugs that treat or prevent cardiovascular disease, concomitant or concomitant diseases, or provide symptomatic relief.

This method provides for intravenous infusion of the pharmaceutical formulations described herein. Any suitable blood vessel may be used for infusion, including peripheral vessels such as those in the forearm of the arm and in the midline that enters the chest. In a preferred embodiment, the pharmaceutical formulation is infused into the endocardium or intravenous blood vessels in the lordosis of the arm of the subject.

In certain embodiments, the method may comprise administering a high or low dose of the Apo A-I Milano: Phosporipid complex pharmaceutical formulation, or a combination thereof. As described below, high dose pharmaceutical formulations over short dosing intervals can safely and effectively reduce atherosclerosis volume in subjects with vessels that are partially or fully occluded. In certain embodiments, pharmaceutical formulations may be administered before, during or after surgical intervention such as PTCA to penetrate occluded blood vessels. In certain embodiments, one or more intermittent doses may be administered to a subject to maintain the patency of previously occluded blood vessels (“maintenance dose”).

The present invention provides a novel method for treating or preventing ischemic reperfusion injury, ie using a pharmaceutical formulation or dose of the Apo A-I Milano complex described herein for the treatment of ischemic reperfusion. Useful doses for such treatments include doses up to 100 mg / kg of the Apo A-I Milano: Phospholipid complex administered to a subject in need thereof. In certain embodiments, the method may comprise administering the pharmaceutical composition of the Apo A-I Milano: Phospholipide complex at a dose of about 1 mg to about 100 mg per kg of body weight of the subject. In a particular embodiment, the method can comprise administering a pharmaceutical composition of the Apo A-I Milano: Phospholipide complex at a dose of about 10 mg / kg to about 50 mg / kg. In a preferred embodiment, the method can comprise administering a pharmaceutical composition of the Apo A-I Milano: Phosporipid complex at a specific dose of about 15 mg / kg. In another preferred embodiment, the method can comprise administering a pharmaceutical composition of the Apo A-I Milano: Phosporipid complex at a specific dose of about 45 mg / kg. The present invention also preferably includes the use of 15 mg / kg alone or in combination with a 45 mg / kg dose. Similarly, a 45 mg / kg dose may be used alone or in combination with a 15 mg / kg dose to treat or prevent ischemic reperfusion injury.

The pharmaceutical formulations provided herein comprise suitable pH, osmolality, tonicity, purity, and sterility of the Apo A-I Milano: Phosporipid complex to enable safe administration to a subject. Pharmaceutical formulations may be formulated for a single single use, or may be formulated to contain antimicrobial excipients as described below to suit multiple doses. In certain embodiments, the pharmaceutical formulation may be contained in a pre-filled sterile syringe or pre-filled sterile bag that may be frozen or refrigerated. In a preferred embodiment, the Apo A-I Milano: Lipid complex is an Apo A-I Milano: Phospolipid complex. In a more preferred embodiment, the Apo A-I Milano: Phospholipid complex is Apo A-I Milano: POPC.

In certain embodiments, pharmaceutical formulations may be included in unit doses or unit-of-use packages. As is known to those skilled in the art, unit dose packages provide one dose delivery of drug to a subject. The method of the present invention provides, for example, a unit dose package of a pharmaceutical formulation comprising 1050 mg of Apo A-I Milano protein per package. For example, 1050 mg of Apo A-I Milano protein is the amount administered to a 70 kg subject to 15 mg / kg of Apo A-I Milano. The unit may be, for example, a single use sterile vial, a pre-filled sterile syringe, a pre-filled sterile bag (ie, a piggyback), and the like.

In certain embodiments, the pharmaceutical formulation may be a use-unit package. As is known to those skilled in the art, the use-unit package is a convenient prescription sized patient unit labeled for direct distribution by a health care provider. The use-unit package contains the pharmaceutical formulation in the amount necessary for a representative treatment interval and duration for a given indication. The method of the present invention comprises, for example, apo AI Milan: phospholipid complex in an amount sufficient to treat an average sized adult male or female intravenously with 15 mg / kg of Apo AI Milano protein once a week for 5 weeks. Provide a use-unit package of a pharmaceutical formulation comprising. Thus, the unit of the use package as described above has 5 doses of the Apo A-I Milano: Phospholipid complex (available in vials or pre-filled syringes). In one embodiment, the use-unit package contains the Apo AI Milano: phospholipide complex in an amount sufficient to treat an average sized adult male or female at a dose of 15 mg / kg or 45 mg / kg once a week for 6 weeks. It includes a pharmaceutical formulation containing. It will be apparent to those skilled in the art that the doses described herein are based on the weight of the subject.

Pharmaceutical formulations may be labeled and include, but are not limited to, instructions contained therein and instructions for use, dosage, interval of administration, duration, indications, contraindications, warnings, precautions, handling and storage instructions, etc. May have attached labeling to identify other information useful to health care workers and subjects in the treatment of acute coronary syndromes.

1 provides the acute effect of a single dose of ETC-216 on serum HDL in normal male subjects;

2 (A) provides a motorized pullback of an IVUS image catheter starting at the distal fiduciary branch (site C) and ending at the proximal branch (site A). . The catheter pathway is shown by angiography;

2 (B) provides a representative cross sectional view of the blood vessel obtained every 0.5 mm until reaching the proximal branch;

2 (C) provides an intermediate cross sectional view of the blood vessel;

2 (D) provides a distal branch in the IVUS image;

3 (A) provides a cross sectional view for analysis;

3 (B) provides an external elastic film (EEM);

3 (C) provides a lumen area;

3 (D) provides a calculation of the maximum atherosclerosis thickness measured by subtracting the cross-sectional area of the lumen from the region of the EEM;

4 (A) provides reference images of blood vessels in patients prior to administration of ETC-216;

4 (B) provides a tracking image of blood vessels in the patient after administration of ETC-216.

5 provides an example of a protocol for treating an isolated rabbit heart with a vehicle or a protein / lipid complex of the invention (ETC-216) before ischemia is expressed;

6 provides creatine kinase activity in coronary venous effluents;

7 provides real-time monitoring of cardiac function collected from vehicle and ETC-216 treated isolated rabbit hearts in a Langendorff Apparatus;

FIG. 8 provides transient changes in left ventricular incidence pressure (LVDP) in rabbit hearts isolated before, during and after 30 minutes of total ischemic arrest and 60 minutes of reperfusion;

FIG. 9 provides transient changes in left ventricular diastolic pressure (LVEDP) in rabbit hearts isolated before, during and after 30 minutes of ischemic arrest and 60 minutes of reperfusion;

FIG. 10 provides transient changes in coronary perfusion pressure in rabbit hearts isolated before, during and after 30 minutes of total ischemic arrest and 60 minutes of reperfusion;

FIG. 11 provides lipid hydroperoxide content in tissue homogenates from vehicle and ETC-216 treated rabbit hearts that caused a total ischemic arrest for 30 minutes, followed by reperfusion for 60 minutes;

12 provides electron microscope images of cardiac muscle samples from vehicle and ETC-216 treated rabbit hearts;

FIG. 13 provides a further protocol of the present invention in which one pretreatment is administered prior to ischemia expression in the acute administration group, and two pretreatments are administered prior to ischemia expression in the chronic administration group;

14 provides a protocol for the measurement of infarct size;

FIG. 15 shows the percent infarct of risk area, percent infarct of left ventricle in rabbits treated once (ie, acute) or twice (ie chronically) with either ETC-216 (100 mg / kg) or an equivalent volume of vehicle. , And% risk area of the left ventricle;

FIG. 16 provides a further protocol of the present invention in which rabbits are pretreated with vehicle (group 1) or 10, 3 or 1 mg / kg ETC-216 (group 2) before ischemia develops;

FIG. 17 shows the percent infarct of risk area, left ventricle measured in rabbits treated once (ie acute treatment) with 10, 3 or 1 mg / kg ETC-216 or equivalent volume of sucrose-mannitol vehicle for each group. % Infarct, and% risk area of the left ventricle;

18 provides for transient changes in lipoprotein nonesterified cholesterol;

FIG. 19 provides a further protocol of the present invention in which a single dose of vehicle or ETC-216 is administered during the last 5 minutes of a 30 minute ischemic period;

FIG. 20 provides the percent infarct of the risk area, percent infarct of the left ventricle, and percent risk area of the left ventricle measured in rabbits.

Justice

As used herein, the following terms have the following meanings:

The term “treat”, “treating” or “treatment” refers to a method of alleviating or eliminating a disease, disorder and / or symptom thereof.

The term “therapeutically effective amount” means an amount of Apo A-I Milano or lipid complex or pharmaceutical formulation thereof sufficient to alleviate to some extent one or more symptoms of the condition or disease to be treated.

The terms "prevent", "preventing" or "prevention" refer to methods of preventing a subject from acquiring a disease, disorder and / or symptoms thereof. In certain embodiments, the terms “prevent”, “preventing” or “prevention” refer to a method of reducing the risk of acquiring a disease, disorder and / or symptom thereof.

The term “prophylactically effective amount” means an amount of Apo A-I Milano or lipid complex or pharmaceutical formulation thereof sufficient to act to prevent the onset or recurrence of one or more symptoms of the condition or disease to be treated.

The term "Acute Coronary Syndrome is the same as instability, Q-wave or non-Q-wave myocardial infarction, or 9th edition replaced by International Classification of Disease 10th Edition (ICD-10). Mean ischemic disease as provided in ICD-9) Myocardial infarction may appear with or without ST-section elevation on the electrocardiogram.

The term "ischemia" refers to ischemia due to mechanical or biologically induced disorders of the blood supply, eg, spasms, thrombosis, stenosis (mainly narrowed or collapsed arteries). The term "myocardial infarction" usually means an inappropriate circulation of blood to the myocardium as a result of coronary artery disease.

The term "ischemic reperfusion" means that the increased amount of oxygenated blood for the tissue is restored.

The term "cardiovascular disease" refers to heart, blood vessel and blood circulation diseases such as myocardial infarction, acute coronary syndrome, atherosclerosis, angina, ischemic reperfusion injury and related diseases.

As used herein, the term "surgical intervention" means a manual, non-pharmacological or surgical method. Surgical intervention may be for diagnostic, radiological, prophylactic or therapeutic purposes.

The term "unstable angina" refers to a change in the frequency, severity, or duration of symptoms of chronic stable angina, such as chest pain or epigastric pain that can spread to the jaw and arm. Chronic stable angina can spread to the subject's jaws and arms, induced by exercise, eating and / or stress, and alleviated by rest without new changes in the frequency or severity of activity required to present symptoms. Pain in the chest.

The term "pharmaceutical formulation" means a composition comprising apo A-I Milano or apo A-I Milano: lipid complex and suitable diluents, carriers, vehicles or excipients suitable for administration to a subject. The term includes, but is not limited to, oral, parenteral and topical compositions as described below.

The term "about" means a relative term indicating an approximate ± 20% of the nominal value it represents. In the field of the present invention, this level of approximation is appropriate unless specifically stated that the value requires a tighter range.

The term "label" means a note written, printed or illustrated on a direct container of a product, such as a note marked on a vial containing a pharmaceutical active.

The term "labeling" refers to a product on or in a container holding such a product, all labels on packaging and other written, printed or illustrated matters, for example a package accompanying or associated with a container of a pharmaceutical active. Means insert, instructional videotape or instructional DVD.

Treatment method

The present invention provides methods and agents for the treatment or prevention of acute coronary syndromes, including unstable angina, ST-segment elevation myocardial infarction and non-Q wave myocardial infarction. Safe and effective dosages for the pharmaceutical formulations described herein have been determined by the applicant for the treatment and prevention of acute coronary syndromes.

Based on what is currently understood, a configuration has been revealed that recognizes a clinical manifestation now called acute coronary syndrome (“ACS”). As noted above, acute coronary syndromes include unstable angina, Q waves, and non-ST-segment elevation myocardial infarction, and are the main cause of morbidity and mortality, especially within the first 24 hours after expression (Schoenhagen et al ., 2000, Circulation 101: 598-603). ACS is an ischemic discomfort that appears without elevation of the ST segment on the ECG. Ischemia often develops into unstable angina, Q-wave and non-Q-wave myocardial infarction (Antman and Braunwald, "Acute Myocardial Infarction" in Heart Disease, A Textbook of Cardiovascular Medicine, 6 ^th edition, vol. 2, Braunwald et al ., eds, 2001, WB Saunders Company, Philadelphia).

The methods and formulations of the present invention provide unique and effective methods for the treatment of atherosclerosis and acute coronary syndromes. In the methods of the present invention, Apo A-I Milano: Phospholipid Complex or pharmaceutical formulation thereof provides a non-surgical treatment that reverses the pathophysiological criteria of the disease rather than providing symptomatic relief. The methods of the present invention provide the administration of Apo A-I Milano, Apo A-I Milano: Phosporipid Complex, or a pharmaceutical formulation thereof, which provides HDL therapy that promotes cholesterol outflow and reverse cholesterol transport and reduces atherosclerosis plaques. Without being bound by any theory, Apo AI Milano, Apo AI Milano: phospholipid complex or pharmaceutical formulations thereof are capable of promoting cholesterol outflow and reverse cholesterol transport, reducing atherosclerosis volume and stabilizing atherosclerosis plaques. It is believed to mimic HDL.

The present invention provides a method for the treatment of acute coronary syndromes. In a preferred embodiment, the Apo AI Milano: phospholipid complex or pharmaceutical formulation thereof has a dose of from about 1 mg (protein) / kg to about 100 mg (protein) / kg, preferably 1 mg (protein) as described below. It can be administered at a dose of / kg to 50 mg (protein) / kg, most preferably 15 mg / kg or 45 mg / kg. Applicants have found that these doses are safe, effective and well accepted by subjects suffering from or at risk of acute coronary syndromes as described in detail herein.

The present invention provides a method for treating or preventing acute coronary syndromes, including alleviating or ameliorating the signs or symptoms of acute coronary syndromes. In certain embodiments, the method is provided for the treatment or reduction of coronary atherosclerosis. In certain embodiments, the method is provided for promoting cholesterol outflow from diseased blood vessels. In certain embodiments, the method is provided to facilitate reverse cholesterol transport. In one embodiment, the method provides reduced atherosclerosis volume in diseased blood vessels. In certain embodiments, the diseased blood vessel is a coronary artery. Atherom volume can be measured by intravascular ultrasound (IVUS) as described herein. In certain embodiments, the method is provided to reduce the total plaque volume of diseased blood vessels. In certain embodiments, the method is provided to reduce the mean maximum plaque thickness in diseased blood vessels. In certain embodiments, the method is provided to reduce the average maximum atherosclerosis thickness. In certain embodiments, this method is provided to reduce plaque volume at a minimum percent plaque area. In certain embodiments, this method is provided to reduce the maximum percent plaque area. In certain embodiments, the method provides an increased mean coronary luminal diameter in the diseased vessel. In certain embodiments, subjects receiving the methods and formulations of the invention may have reduced angiographic lesions compared to subjects not receiving the methods and formulations of the invention. In certain embodiments, the method provides for regression of an existing lesion. In certain embodiments, the methods and pharmaceutical formulations are provided to obtain patency of occluded blood vessels or maintain patency of occluded blood vessels.

In certain embodiments, the dose of Apo A-I Milano: Phospholipid complex of about 15 mg / kg to about 45 mg / kg provides an average percent reduction in atherosclerosis of about 2%, about 1%, or about 0.05%. In certain embodiments, the dose of apo AI Milano: phospholipide complex of about 15 mg / kg to about 45 mg / kg provides a reduction in atherosclerosis of about 20 Hz, about 15 Hz, about 10 Hz or about 5 Hz. do. In certain embodiments, a dose of Apo A-I Milano: Phospholipide complex of about 15 mg / kg to about 45 mg / kg provides a reduction in average maximum atherosclerosis thickness from about -0.039 mm to -0.044 mm.

In one embodiment, the method provides for the treatment of acute coronary syndromes in a subject having signs or symptoms of acute coronary syndromes. In one embodiment, the subject has signs and / or symptoms of myocardial ischemia, such as pain in the chest, jaw, arm or upper abdomen, palpitations, shortness of breath, sweating, nausea and / or vomiting. Can be represented. In another embodiment, the method is accompanied by changes in electrocardiogram ("ECG" or "EKG"), such as T wave changes such as ST fragment elevation, inversion, and an increase in creatine kinase fraction, troponin I or C-reactive protein. Treatment of acute coronary syndromes is provided in a subject exhibiting signs and symptoms of acute coronary syndromes.

In one embodiment, the method provides for the prevention of acute coronary syndromes in a subject at risk of developing acute coronary syndromes. Subjects at risk include subjects of various ages (eg, 18-24, about 25, about 30, about 40, about 50, about 60, about 70, about 80, or about 90 years old), family history of cardiovascular disease, or cardiovascular Subjects with genetic predisposition to the disease, diabetes, hypertension, multiple-vessel or left-mainstem disease, or subjects who have previously had a myocardial infarction.

It is understood by those skilled in the art that the actual dose of the formulation of the present invention may vary depending on the subject's height, weight, age, severity of the disease, the presence of accompanying medical conditions, and the like. For example, elderly subjects with impaired renal or hepatic function may be treated with a dose of the Apo AI Milan: Lipid Complex in a lower range of about 1 mg / kg dose (eg, 0.8 mg / kg or 0.9 mg / kg). Can be. Subjects with good kidney and liver function and who are obese and have severe acute coronary syndromes may, for example, have upper ranges of about 100 mg / kg doses (eg, 120 mg / kg, 119 mg / kg, 118 mg / kg). Kg, 115 mg / kg, etc.), may be treated with a dose of Apo AI Milan: Lipid Complex. Dosages of the formulations described herein have been shown to be effective in achieving the intended purpose. These doses obtain a range of circulating concentrations that include an effective amount with an acceptable risk for a beneficial profile.

The present invention provides a method of treating or preventing acute coronary syndrome with a dosage dosing schedule sufficient to treat acute coronary syndrome in a subject in need thereof. In certain embodiments, the Apo AI Milano: phospholipide complex or pharmaceutical formulation thereof as described below is approximately daily, approximately every other day, approximately every three days, approximately every four days, approximately every five days, Approximately every 6 days, approximately every 7 days, approximately every 8-10 days, or approximately every 11-14 days. This period is also called the dosing interval or interval. In certain embodiments, the dosing interval may be monthly, every 6 months, every 12 months, every 18 months or every 24 months. In a preferred embodiment, Apo A-I Milano, Lipid Complex or a pharmaceutical formulation thereof can be administered approximately every seven days. In certain embodiments, administration of the Apo A-I Milano: Phospholipide complex or pharmaceutical formulation thereof may be a single administration. In certain embodiments, the administration is continued for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7-12 weeks, about 13-24 weeks, about 52 weeks, or Or during the subject's survival. This period is also called the dosing period, treatment period or period. Thus, the dosage administration schedule may be, for example, a pharmaceutical formulation of the Apo A-I Milano: phospholipide complex administered approximately every seven days for about six weeks. In certain embodiments, the dosing interval may continue intermittently after about 52 weeks. For example, the subject may be treated once a week for about 52 weeks, then about 3 to about 4 times over the next year. In certain preferred embodiments, administration of the pharmaceutical formulation containing the Apo A-I Milano: phospholipid complex may occur approximately every seven days for about five weeks. Other dosage administration schedules using various dosage intervals and durations are also included within the scope of the present invention as described herein.

The dose of Apo A-I Milano: phospholipide complex or a pharmaceutical formulation thereof may vary over the duration of treatment. For example, a subject may be treated once a week for three weeks with a pharmaceutical formulation of 45 mg / kg of Apo AI Milano: phospholipide complex, followed by 15 mg / kg of Apo AI Milano: phospholipide complex. The agent may be treated once every four months or once a year during the subject's survival. Such intermittent doses may be administered to maintain the patency of the blood vessels. Also included within the scope of the invention are intermittent doses administered during the subject's survival to maintain reduced atherosclerosis and increased vascular lumen.

The methods and formulations of the invention can be used with surgical intervention, ie before, during or after surgery. Surgical interventions may include angioplasty, intravascular ultrasound, coronary artery bypass graft (CABG), coronary angioplasty, vascular stent implantation, percutaneous coronary intervention (PCI) and / or stabilization of plaque. In certain embodiments, the method provides for administering an Apo AI Milano: phospholipid complex or a pharmaceutical formulation thereof before or after surgical intervention to open a blocked vessel or reduce atherosclerotic plaque in the vessel. do. Surgical intervention refers to a manual, non-pharmacological or surgical method used to diagnose, image (radiology), prevent or treat a disease or condition. For example, intravascular ultrasonography (IVUS) and coronary angiography are methods that can provide a quantitative assessment of plaque load (diagnostic purpose), and angiography can provide an image of blood vessels (radiographic purpose). Angioplasty may open the occluded blood vessel (for therapeutic purposes). These are all included as surgical interventions used in the present invention.

Apolipoprotein  A-I Milano

In one aspect, the present invention provides methods and formulations for treating, reducing or preventing damage from acute coronary syndromes by administering formulations comprising Apo A-I Milan. In certain embodiments, Apo A-I Milano may form a complex with lipids.

Human Apo AI Milan is a natural variant of Apo AI (Weisgraber et al . 1980, J. Clin . Invest . 66: 901-907). In Apo AI Milan the amino acid arginine (Arg173) is substituted by the amino acid cysteine (Cys173). Because Apo AI Milan contains one cysteine residue per polypeptide chain, it may exist in the form of a monomer, homodimer or heterodimer (see US Pat. No. 5,876,968, incorporated herein in its entirety by this nomenclature). . These forms are chemically interchangeable, and the term Apo AI Milano does not distinguish between them. The variant form at the DNA level is the result of replacing C with T in the gene sequence, ie, codon CGC with TGC, which translates into cysteine (Cys) instead of arginine (Arg) at the amino acid position 173.

In certain embodiments, Apo A-I Milano may be a variant or a conservatively substituted Apo A-I Milano. Conservative substitutions mean that certain amino acid residues of Apo A-I Milano can be substituted with other amino acid residues without significantly detrimental effect on the activity of the protein. Accordingly, modified or substituted forms of Apo A-I Milano, wherein at least one defined amino acid residue in the structure is replaced with another amino acid residue, are also contemplated by the present invention. For the purpose of measuring conservative amino acid substitutions, amino acids may conveniently be classified into two main categories, hydrophilic and hydrophobic, mainly depending on the physical-chemical properties of the amino acid chain. These two main categories can be further divided into subcategories that more clearly define the characteristics of the amino acid chain. For example, the class of hydrophilic amino acids can be further subdivided into acidic, basic and polar amino acids. The class of hydrophobic amino acids may be further subdivided into nonpolar and aromatic amino acids. Definitions of the various categories of amino acids that define structure (I) are as follows.

"Hydrophilic amino acid" means an amino acid that exhibits hydrophobicity below 0 according to the normalized consensus hydrophobicity scale of Eisenberg et al., 1984, J. Mol. Biol. 179: 125-142. do. Genetically encoded hydrophilic amino acids include Thr (T), Ser (S), His (H), Glu (E), Asn (N), Gln (Q), Asp (D), Lys (K) and Arg (R ) Is included.

"Acid amino acid" means a hydrophilic amino acid having a side chain pK value of less than 7. Acidic amino acids typically have negatively charged side chains at physiological pH due to the loss of hydrogen ions. Acidically encoded acidic amino acids include Glu (E) and Asp (D).

"Base amino acid" means a hydrophilic amino acid having a side chain pK value greater than 7. Basic amino acids typically have positively charged side chains at physiological pH due to binding to hydronium ions. Genetically encoded basic amino acids include His (H), Arg (R) and Lys (K).

"Polar amino acid" means a hydrophilic amino acid having a side chain that is uncharged at physiological pH, but has at least one bond in which pairs of electrons commonly held by two atoms are pulled closer by one of the atoms. Genetically encoded polar amino acids include Asn (N), Gln (Q) Ser (S) and Thr (T).

"Hydrophobic amino acid" means an amino acid that exhibits hydrophobicity greater than zero according to the standardized consensus hydrophobicity scale of Eisenberg, 1984, J. Mol. Biol. 179: 125-142. Hydrophobic amino acids encoded by the gene include Pro (P), Ile (I), Phe (F), Val (V), Leu (L), Trp (W), Met (M), Ala (A), Gly (G ) And Tyr (Y).

"Aromatic amino acid" means a hydrophobic amino acid with side chains having at least one aromatic or heteroaromatic ring. Aromatic or heteroaromatic rings include --OH, --SH, --CN, --F, --Cl, --Br, --I, --NO ₂ , --NO, --NH ₂ , --NHR , --NRR, --C (O) R, --C (O) OH, --C (O) OR, --C (O) NH ₂ , --C (O) NHR, --C ( O) NRR, and the like may contain one or more substituents, wherein each R is independently (C ₁ -C ₆ ) alkyl, substituted (C ₁ -C ₆ ) alkyl, (C ₁ -C ₆ ) Alkenyl, substituted (C ₁ -C ₆ ) alkenyl, (C ₁ -C ₆ ) alkynyl, substituted (C ₁ -C ₆ ) alkynyl, (C ₅ -C ₂₀ ) aryl, substituted ( C ₅ -C ₂₀ ) aryl, (C ₆ -C ₂₆ ) alkaryl, substituted (C ₆ -C ₂₆ ) alkaryl, 5-20 membered heteroaryl, substituted 5-20 membered heteroaryl, 6- 26 membered alkheteroaryl or substituted 6-26 membered alkheteroaryl. Genetically encoded aromatic amino acids include Phe (F), Tyr (Y) and Trp (W).

"Non-polar amino acids" are uncharged at physiological pH and have side chains in which pairs of electrons commonly held by two atoms generally have an equal pull by each of the two atoms (ie, the side chains are not polar) It means a hydrophobic amino acid having. Genetically encoded nonpolar amino acids include Leu (L), Val (V), Ile (I), Met (M), Gly (G) and Ala (A).

"Alphaic amino acid" means a hydrophobic amino acid having an aliphatic hydrocarbon side chain. Aliphatic amino acids encoded by the gene include Ala (A), Val (V), Leu (L) and Ile (I).

The amino acid residue Cys (C) is a unique one capable of forming a disulfide bridge with other Cys (C) residues or other sulfanyl-containing amino acids. The ability of Cys (C) residues (and other amino acids with --SH containing side chains) present in the peptide in reduced free-SH or oxidized disulfide-bridged form is such that the Cys (C) residues are pure hydrophobic to the peptide or Affects whether or not it provides hydrophilic features. Cys (C) exhibits a hydrophobicity of 0.29 according to the Eisenberg, 1984, reference standardized consensus scale, but for the purposes of the present invention Cys (C) is used despite the general classification defined above. It is understood to be classified into the category of polar hydrophilic amino acids.

As will be appreciated by those skilled in the art, the above-defined categories are not mutually exclusive. Thus, amino acids having side chains that exhibit two or more physico-chemical properties can be included in multiple categories. For example, amino acid side chains having aromatic moieties further substituted with polar substituents such as Tyr (Y) can exhibit both aromatic hydrophobic properties and polar or hydrophilic properties and thus can be included in both aromatic and polar categories. Appropriate categorization of amino acids will be apparent to those skilled in the art, in particular in light of the detailed description provided herein. In certain embodiments, the substituted or modified form of Apo A-I Milano is not Apo A-I.

Apo AI Milan used in the present invention can be obtained from any source available. For example, Apo AI Milan can be recombinant, synthetic, semisynthetic or purified Apo AI Milan. In a preferred embodiment, Apo AI Milano uses yeast or E. coli as described in US Pat. No. 5,721,114 and EP 0 469 017 and EP 0 267 703, which are hereby incorporated by reference in their entirety. It may be a recombinant protein (rApo AI Milano) expressed in E. coli . Methods of obtaining Apo AI Milan used by the present invention are well known in the art. For example, Apo AI Milan can be used for, for example, denitrification, reduction, denaturation and gel-filtration chromatography, ion-exchange chromatography, hydrophobicity, for example, phenyl sepharose interaction of density gradient centrifugation followed by lipoprotein. Recombinant DNA techniques that can be isolated from plasma by functional chromatography or immunoaffinity chromatography, or that are known synthetically, semisynthetically, or to those skilled in the art, and then to those skilled in the art. Can be produced using purification methods (see, eg, US Pat. Nos. 6,107,467; 6,559,284; 6,423,830; 6,090,921; 5,834,596; 5,990,081; 6,506,879, Mulugeta et. al ., 1998, J. Chromatogr . 798 (1-2): 83-90; Chung et al ., 1980, J. Lipid Res . 21 (3): 284-91; Cheung et al ., 1987, J. Lipid Res . 28 (8): 913-29; Persson, et al ., 1998, J. Chromatogr . 711: 97-109; U.S. Patents 5,059,528, 5,834,596, 5,876,968 and 5,721,114; And PCT publications WO 86/04920 and WO 87/02062.

If Apo A-I Milano is obtained from a natural source, it can be obtained from any species of animal source. In certain embodiments, Apo A-I Milano can be obtained from a mammalian source. In certain embodiments, Apo A-I Milano can be obtained from a human source. In a preferred embodiment of the invention, Apo A-I Milan is derived from the same species as the subject to which Apo A-I Milan is administered. In a preferred embodiment, Apo A-I Milano is a recombinant Apo A-I Milano protein.

Method of administration

Apo AI Milano: phospholipide complexes or pharmaceutical formulations thereof may be administered by any suitable route known to those skilled in the art, which ensures bioavailability in the circulation. Any route of administration that provides a therapeutically effective amount of the formulation of the invention can be used. The route of administration may be indicated by the type of pharmaceutical formulation. For example, injectable preparations include intravenous (IV), intramuscular (IM), intradermal, subcutaneous (SC), endovascular, intraarterial, intracardiac, intraarticular and intraperitoneal (IP) injections, It may be administered parenterally (but not limited to these) (see, for example, Robinson et. al ., 1989, In: Pharmacotherapy: A Pathophysiologic Approach, Ch. 2, pp. 15-34; This full name is incorporated herein in its entirety).

In a preferred embodiment, the Apo A-I Milano: phospholipide complex or pharmaceutical formulation thereof can be administered parenterally. In a more preferred embodiment, parenteral administration is intravenous. Intravenous administration can be, for example, by bolus administered over about 2-3 minutes, or by continuous infusion, for example by using a pump over about 1 hour, or at about 24 hours. Can be injected continuously over. In a preferred embodiment, the infusion can be over about 1 to about 3 hours.

This method provides for intravenous infusion of the pharmaceutical formulations described herein. Any suitable blood vessel, including peripheral blood vessels, such as the forearm of the arm and the central line entering the chest, can be used as the site of infusion. In a preferred embodiment, the pharmaceutical formulation is infused into the endocardium or intravenous blood vessels in the lordosis of the arm of the subject.

In certain embodiments, administration can be performed by a mechanical pump or delivery device, such as a pericardial delivery device (PerDUCER®) or a cardiopulmonary bypass device.

Lipid  Complex

In certain embodiments, the methods of the present invention comprise administering a lipid complex of Apo A-I Milano. In certain embodiments, the present invention provides a pharmaceutical formulation of the Apo A-I Milano: Phospholipide complex. Efficacy can be enhanced by making Apo A-I Milano and Lipid into a complex. In general, the lipid is mixed with Apo A-I Milano prior to administration. Apo AI Milano and Lipid can be mixed in an aqueous solution at an appropriate ratio and complexed by methods known in the art, including freeze-drying, detergent solubilization followed by dialysis, microfluidization, sonication, and homogenization. Can be formed. Composite efficiency can be optimized, for example, by varying the pressure, ultrasonic frequency or detergent concentration. An example of a detergent commonly used to prepare the Apo A-I Milano: Phospholipide complex is sodium cholate.

In some cases, it is desirable to administer Apo A-I Milano alone, essentially without lipids, to treat or prevent acute coronary syndrome. In a preferred embodiment, the Apo A-I Milano: Lipid Complex is administered to a subject in need thereof.

In one embodiment, the Apo A-I Milano: phospholipid complex may be present in solution with an appropriate pharmaceutical diluent. In another embodiment, the freeze-dried or lyophilized formulation of the Apo A-I Milano: phospholipid complex may be hydrated or re-formed with an appropriate pharmaceutical diluent prior to administration. In another embodiment, the Apo A-I Milano: Lipid complex can be a frozen preparation that thaws until a homogeneous solution is obtained prior to administration to a subject in need thereof.

Lipids may be any suitable lipids known to those skilled in the art. Stearylamine, dodecylamine, acetyl palmitate, (1,3) -D-mannosyl- (1,3) diglycerides, aminophenylglycosides, 3-cholesteryl-6 '-(glycosylthio Hexyl Ether Glycolipid, N- (2,3-di (9- (Z) -octadecenyloxy))-prop-1-yl-N, N, N-trimethylammonium chloride and fatty acid amide Non-phosphorus containing lipids that are not limited to these may be used. In a preferred embodiment, the lipid is phospholipid.

Phospholipids can be obtained from any source known to those skilled in the art. For example, phospholipids can be obtained from commercial sources, natural sources, or by synthetic or semisynthetic methods known to those skilled in the art (Mel'nichuk et al. al ., 1987, Ukr. Biokhim. Zh . 59 (6): 75-7; Mel'nichuk et al . , 1987, Ukr . Biokhim . Zh . 59 (5): 66-70; Ramesh et al . , 1979, J. Am . Oil Chem . Soc . 56 (5): 585-7; Patel and Sparrow, 1978, J. Chromatogr . 150 (2): 542-7; Kaduce et al . , 1983, J. Lipid Res . 24 (10): 1398-403; Schlueter et al . , 2003, Org . Lett . 5 (3): 255-7; Tsuji et al . , 2002, Nippon Yakurigaku Zasshi 120 (1): 67P-69P).

In a preferred embodiment, the lipid may be a phospholipid. Phospholipids can be any phospholipid known to those skilled in the art. For example, phospholipids are small alkyl chain phospholipids, phosphatidylcholine, egg phosphatidylcholine, soy phosphatidylcholine, dipalmitoylphosphatidylcholine, soybean phosphatidylglycerol, egg phosphatidylglycerol, distearoylphosphatidylglycerol, dimyristoylphosphatidylcholine, disi Theoylylphosphatidylcholine, dilaurylphosphatidylcholine, 1-myristoyl-2-palmitoylphosphatidylcholine, 1-palmitoyl-2-myristoylphosphatidylcholine, 1-palmitoyl-2-stearoylphosphatidylcholine, 1-stearoyl 2-palmitoylphosphatidylcholine, dioleoylphosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylcholine, 1-oleoyl-2-palmitylphosphatidylcholine, dioleoylphosphatidylethanolamine, dilauroylphosphatidylglycerol, phosphatidylserine Phosphatidylethanolamine, phosphatidyl inositol, phosphatidylglycerol, diphospha Thidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dioleoylphosphatidylglycerol, phosphatidic acid, dimyristoylphosphatidic acid, dipalmitoylphosphatidine acid, dimyri Stoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylserine, dipalmitoylphosphatidylserine, brain phosphatidylserine, sphingomyelin, sphingolipid, brain sphingomyelin, dipalmitoyl spingo Myelin, distearoylsphingomyelin, galactocerebroside, ganglioside, cerebroside, phosphatidylglycerol, phosphatidic acid, lyso lecithin, lysophosphatidylethanolamine, cephalin, cardiolipin, disetyl Phosphates, distearoyl-phosphatidylethanolamine and cholesterol and derivatives thereof.

Phospholipids may also be derivatives or analogs of any of the above phospholipids. In certain embodiments, the Apo A-I Milano: Phospholipid complex may comprise a combination of two or more phospholipids.

In a preferred embodiment, the methods of the present invention provide for the administration of the Apo AI Milano: phospholipid complex. In a more preferred embodiment, the lipid is phospholipid, preferably 1-palmitoyl-2-oleoyl phosphatidylcholine (“POPC”) or (“1-palmitoyl-2-oleoyl- sn -glycero-3- Phosphocholine "). In even more preferred embodiments, the Apo AI Milano: POPC complex is in a ratio of about 1: 1 by weight of Apo AI Milano: POPC. In the most preferred embodiment, the Apo AI Milano: POPC complex is a pharmaceutical formulation. Apo AI Milano: POPC complex is called ETC-216.

The complex consisting of Apo A-I Milano and Lipid may contain any amount of Apo A-I Milano, and any amount of lipid, preferably phospholipid, effective to treat or prevent acute coronary syndrome. In certain embodiments, Aper A-I Milan may consist of a complex of Apo A-I Milano and phospholipid in a ratio of about 1 to about 1 by weight. However, Apo AI Milan is about 100: 1, about 10: 1, about 5: 1, about 3: 1, about 2: 1, about 1: 1, about 1: 2, about 1: 3, about 1: 5 , About 1:10 and about 1: 100 (weight of protein / weight of lipid), and other proportions of phospholipid to Apo AI Milan. The most uniform population of weight ratios from about 1: 0.5 to about 1: 3 (weight of protein / weight of lipid), more preferably from about 1: 0.8 to about 1: 1.2 (weight of protein / weight of lipid) It is preferred for the purpose of producing, producing stable and reproducible batches. In an even more preferred embodiment, the ratio of Apo A-I Milano to phospholipid is 1: 0.95 (weight of protein / weight of lipid).

Additional lipids suitable for use in the methods of the present invention are well known to those skilled in the art and are mentioned in a variety of well known materials (eg, McCutcheon's Detergents and Emulsifiers and McCutcheon's Functional Materials, Allured Publishing Co., Ridgewood, NJ; both of which are incorporated herein by this name). In general, the lipid is preferably liquid-crystalline at 37 ° C, 35 ° C, or 32 ° C. Lipids in the liquid-crystalline state generally receive cholesterol more efficiently than lipids in the gel state. Because subjects generally have a core temperature of about 37 ° C., liquids that are liquid-crystalline at 37 ° C. are generally liquid-crystalline during treatment.

Lipid  Complex of  Produce

Apo A-I Milano: Lipid complexes can be prepared by any method known to those skilled in the art. In some cases, it is desirable to mix Lipid with Apo A-I Milano prior to administration. Lipids may be in solution or in the form of liposomes or emulsions formed using standard techniques such as homogenization, sonication or extrusion. Sonication is generally performed using tip Sonyfire, such as Branson tip sonifier, in an ice bath. In general, the suspension is subjected to several sonication cycles. Extrusion can be performed by a biomembrane extruder such as a Lipex Biomembrane Extruder® (Lipex Biomembrane Extruder, Inc. Vancouver, Canada). The defined pore size in the extrusion filter can produce a monolamellar liposomal vesicle of defined size. Pyrosomes can also be obtained through asymmetric ceramic filters, such as Ceraflow Microfilter®, available commercially from Norton Company, Worcester Mass., Or through polycarbonate filters or those skilled in the art. It can be formed through other types of polymerized materials (ie plastics) known to the expert.

Apo AI Milano: Lipid complexes can be prepared in a variety of forms, including but not limited to vesicles, liposomes or proteoliposomes. Various methods well known to those skilled in the art can be used to prepare the Apo AI Milano: Lipid complex. Many available techniques for preparing liposomes or proteoliposomes can be used. For example, Apo AI Milan can be co-ultrasounded with an appropriate lipid (using a bath or probe sonicator) to form a lipid complex. In certain embodiments, Apo AI Milan may be combined with preformed lipid vesicles to induce spontaneous formation of the apolipoprotein: lipid complex. In another embodiment, Apo AI Milan may also be prepared by a detergent dialysis method, for example, by dialysis of a mixture of detergents such as Apo AI Milan, Lipid, and Cholate to remove the detergent, re-form to lipid Complexes may also be prepared (see Jonas et. al ., 1986, Methods Enzymol . 128, 553-82).

In another embodiment, the lipid complex can be prepared by co-freezing as described in US Pat. Nos. 6,287,590 and 6,455,088, which are incorporated herein in their entirety via this name. Other methods are described, for example, in US Pat. Nos. 6,004,925, 6,037,323 and 6,046,166, which are hereby incorporated by reference in their entirety. Other methods of preparing the Apo A-I Milano: Lipid complex will be apparent to those skilled in the art.

In a preferred embodiment, the lipid complex can be prepared by homogenization. In a preferred embodiment, the preparation of the Apo A-I Milano: Lipid Complex begins by diluting the recombinant Apo A-I Milano with a water for injection to a concentration of 15 mg / ml in solution. Sodium phosphate is added at a final concentration of 9-15 mM phosphate and the pH is adjusted to between about 7.0 and about 7.8. Mannitol is added to obtain a concentration of about 0.8% to about 1% mannitol (w / v). POPC is then added to obtain a mixture of about 1: 0.95 (wt protein / wt lipid) of Apo A-I Milano for POPC. The mixture is stirred at 5000 rpm for about 20 minutes using an overhead propeller and Ultra Turrax while maintaining a temperature between 37 ° C. and 43 ° C. The temperature is maintained between 32 ° C. and 43 ° C. using in-line heat exchangers (Avestin, Inc.) while the feed vessel is continuously stirred at 300 rpm. Homogenization for the first 30 minutes is carried out at 50 MPa (7250 psi), after which the pressure is 80-120 until the test during the preparation by gel permeation chromatography shows> 70% AUC% between protein standards. Maintain at MPa (11600-17400 psi). The complex may also be prepared in 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml and 14 mg / ml formulations, where the weight is the weight of the protein.

Combination therapy

Apo A-I Milano or lipid complexes or pharmaceutical formulations thereof may be used alone or in combination with other interventions in the methods of the invention. Such therapies include, but are not limited to, simultaneous or sequential administration of other drugs.

In certain embodiments, the methods and formulations of the present invention can be used in combination with other drugs to obtain the methods provided herein. Co-administration with another drug can treat, prevent or ameliorate the accompanying disease, for example, an antiarrhythmic agent administered treats a coexisting arrhythmia. In certain embodiments, a method of co-administering a drug is provided to treat or prevent pain associated with acute coronary syndromes.

As mentioned above, conventional therapies for ischemic attacks including myocardial infarction, angina and acute coronary syndromes have not been targeted for the protopathology of collapsed or unstable atherosclerotic plaques. For example, subjects exhibiting cardiac chest discomfort or other ischemic symptoms include anticoagulants or antithrombotic agents such as aspirin, clopidogrel, heparin, epitifibatide, or abscimab Emergency percutaneous coronary intervention (PCI) can be prepared. Beta-adrenergic blockers (β-blockers) may be administered to reduce heart rate. Administration of angiotensin converting enzyme inhibitors (ACE inhibitors) is recommended to subjects with concomitant congestive heart failure (CHF) or left ventricular (LV) dysfunction. In subjects who have revived from sudden cardiac death, administration of amiodarone is recommended. Oxygen is often supplied to the subject, usually by a mask or nasal cannula, and the vital sign, including oxygen saturation by pulse oxumetry, is closely monitored. For long-term treatment, statins of HMGCoA reductase inhibitors are often administered to the subject to reduce LDC levels.

For example, Apo AI Milano or Lipid Complex or pharmaceutical formulations thereof may include alphabeta adrenergic antagonists, antiadrenergic agonists, alpha-1 adrenergic antagonists, beta adrenergic antagonists, AMP kinase activators, angiotensin converting enzymes (ACE) inhibitors, Angiotensin II receptor antagonists, calcium channel blockers, antiarrhythmics, vasodilators, nitrates, vasoconstrictors, inotropic agents, diuretics, anticoagulants, antiplatelet aggregates, thrombolytics, antidiabetic agents, antioxidants, anti-inflammatory agents, bile acids Bile acid sequestrants, statins, cholesterol ester transfer protein (CETP) inhibitors, cholesterol reducing agents / lipid regulators, arachidonic acid conversion blocking drugs, estrogen replacement therapy, fatty acid homologs, fatty acid synthesis inhibitors, fibrate, histidine, nicotinic acid derivatives, Peroxysome proliferation activator receptor agonists or antagonists, Fatty acid oxidation inhibitors, thalidomide or thiazolidinediones ( Drug Facts and Comparisons , updated monthly, January 2003, Wolters Kluwer Company, St. Louis, MO; Physicians Desk Reference (56 ^th edition, 2002) Medical Economics) may be administered with other pharmaceutically active agents including, but not limited to.

Other drugs that may be added alone or in combination to Apo AI Milano, Lipid Complex, or pharmaceutical formulations thereof, or which may enhance their beneficial properties, include, but are not limited to: Alpha / Beta adrenergic antagonists (“β-blockers”), for example carvediols (Coreg®); Labetalol HCl (Normodyne®); Antiadrenergic agents such as guanadrel (Hylorel®); Guanetidine (Ismelin®); Leisure pin, clonidine (Catapres® and Catapress-TTS®); Guanfacin (Tenex®); Guanabenz (Wytensin®); Methyldopa and methyldopate (Aldomet®); Alpha-1 adrenergic antagonists such as doxazosin (Cardura®); Prazosin (Minipress®); Tetrazosin (Hytrin®); And phentolamine (Regitine®); Beta adrenergic antagonists, such as sotarol (Betapace AF® and Betatace®); Timolol (Blocadren®); Propranolol (InderalLA® and Inderal®); Betaxolol (Kerlone®); Acebutorol (Sectral®); Atenolol (Tenormin®); Metoprolol (Lopressor® and Toprol-XL®); Arsenoprolol (Zebata®); Carteolol (Cartrol®); Esmolol (Brevibloc®); Naldorol (Corgard®); Fenbutorol (Levatol®); And pindorol (Visken®); AMP kinase activators such as ESP 31015, (ETC-1001); ESP 31084, ESP 31085, ESP 15228, ESP 55016 and ESP 24232; Gemcaben (PD 72953 and CI-1027); And Medica 16; Angiotensin converting enzyme (ACE) inhibitors such as quinapril (accupril®)); Benazepril (Lotensin®); Captopril (Capoten®); Enalapril (Vasotec®); Ramipril (Altace®); Fosinopril (Monopril®); Moexipril (Univasc®); Lisinopril (Prinivil®) and Zestril®); Transdorapril (Mavik®), perindopril (Aceon®); And angiotension II receptor antagonists such as candesaartan (Atacand®); Irbesartan (Avapro®); Losartan (Cozaar®); Valsartan (Diovan®); Telmisartan (Micardis®); Eprosartan (Tevetan®); And olmesartan (Benicar®); Calcium channel blockers such as nifedipine (Adalat®, Adalat CC®, Procardia® and Procardia XL®); Verapamil (Calan®, Callan SR®, Covera-HS®, Isoptin SR®, Verelan® and Bereran PM®); Diltiazem (Cardizem®, CardizemCD® and Tiazac®); Nimodipine (Nimotop®); Amlodipine (Norvasc®); Felodipine (Plendil®); Nisoldipine (Sular®); Bepridil (Vascor®); Isradipine (DynaCirc®); And nicardipine (Cardene®); Antiarrhythmic agents such as various quinidines; Procaineamide (Pronestyl® and Procan®); Lidocaine (Xylocaine®); Mexyltine (Mexitil®); Tocanide (Tonocard®); Flecainide (Tambocor®); Propafenone (Rythmol®) , moricygin (ethmozin®)); Ibutylide (Covert®); Disopyramid (Norpace®); Bretyllium (Bretylol®); Amiodarone (Cordarone®); Adenosine (Adenocard®); Dofetilide (Tikosyn®); And digoxin (Lanoxin®); Vasodilators such as diazoxide (Hyperstat IV®); Hydralazine (Apresoline®); Fenoldopam (Corolpam®); Minoxidil (Loniten®); And nitroprusside (Nipride®); Nitrates such as isosorbide dinitrate (Isordil® and Sorbitrate®); Isosorbide mononitrate (Imdur®, Ismo® and Monoket®); Nitroglycerin paste (Nitrol®); Various nitroglycerin patches; Nitroglycerin SL (Nitrostat®), Nitrolingual spray; And nitroglycerin IV (Tridil®); Vasoconstrictors such as norepinephrine (Levophed®); And phenylephrine (Neo-Synephrine®); Transformers such as amlinone (Inocor®); Dopamine (Intropine®); Dobutamine (Dobutrex®); Epinephrine (Adrenalin®); Isoproterenol (Isuprel®), milinon (Primacor®); Diuretics such as spironolactone (Aldactone®); Torsamide (Demadex®); Hydroflumetiazide (Diucardin®); Chlorothiazide (diuril®)); Ethacrynic acid (Edecrin®); Hydrochlorothiazide (hydroDIURIL® and Microzide®); Amylolide (Midamor®); Chlorthalidone (Thalitone® and Hygroton®); Bometanide (Bumex®); Purosemide (Lasix®); Indafamid (Lozol®); Metolazone (Zaroxolyn®); Triamterene (Dyrenium ®); And combinations of triamterene and hydrochlorothiazide (Dyazide® and Maxzide®); Antithrombotic / anticoagulant / antiplatelet agents such as vivalirudin (Angiomax®); Lepirudine (Refludan®); Various heparin; Danaparoid (Orgaran®); Various low molecular weight heparins; Dalteparin (Fragmin®); Enoxaprine (Lovenox®); Tinzaparin (Innohep®); Warfarin (Coumadin®); Dicumarol (Dicoumarol®); Anicindione (Miradone®); aspirin; Argatroban (Argatroban®); Absiazimab (Reopro®); Eptipibatide (Integrilin®); Tyropiban (Agragrastat®); Clopidogrel (Plavix®); Ticlopidine (Ticlid®); And dipyridamole (Persantine®); Thrombolytics such as alteplace (Activase®); Tissue plasminogen activator (TPA) (Activase®); Annithreplaces, APSAC (Eminase®); Leteplace, rPA (Retavasae®); Steptokinase, SK (Streptase®); Urokinase (Abbokinase®); antidiabetic agents such as metformin (Glucophage®); glipizide (Glucotrol®); chloropropamide (Diabinese) ®)); Acetohexamide (Dymelor®); Tolazamide (Tolinase®); Glymepride (Amaryl®); Glylide (DiaBeta® ) And Micronase®); Acarbose (Precose®); Miglytol (Glyset®); Repaplinide (Prandin®); Nateglinide (Star Starlix®); rosiglitazone (Avandia®); and pioglitazone (Actos®); antioxidants and anti-inflammatory agents; bile acid sequestrants such as cholestyramine (LoCholest®) , Prevalite® and Questran®; cholestipol (Colestid®); and Recebelam (Welchol®); statins (drugs that inhibit HMGCoA reductase), for example, lovastatin (Crestor®); fluvastatin (Lescol®); atorvastatin (lipitor ( Lipitor®)); lovastatin (Mevacor®); pravastatin (Pravachol®); and simvastatin (Zocor®); CETP inhibitors; drugs that block arachidonic acid conversion: estrogen replacement therapy; Fatty acid homologues such as PD 72953, MEDICA 16, ESP 24232 and ESP 31015; fatty acid synthesis inhibitors; fatty acid antioxidants, ranolazine (Ranexa®); fibrate, for example clofibrate ( Atromid-S®); gemfibrozil (Lopid®); micronized fenofibrate capsules (Tricor®); bezafibrate and cypropibrate; Histidine; Nicotinic acid derivatives such as niacin sustained release tablets (Niaspan®); Peroxysome growth activator receptor agonists and antagonists; Thalidomide (Thalomid®) and U.S. Pat.Nos. 6,459,003 and 6,506,799 and U.S. Patent Publication Nos. 20030022865, 20030018013, 20020077316, and 20030078239, the contents of which are incorporated herein by reference in their entirety. Compound.

Other drugs that may be added alone or in combination to Apo AI Milan or Lipid Complex or pharmaceutical formulations thereof, or which may enhance their beneficial properties, include, for example, antiproliferative drugs such as paclitaxel and topotecan ( Brehm et al . 2001, Biochemical Pharmacology , 61 (1): 119-127) and anti-inflammatory drugs such as steroidal and non-steroidal anti-inflammatory agents, including cyclooxygenase-2 (COX-2) inhibitors.

Pharmaceutical preparations

In one aspect, the present invention provides an agent for treating, reducing or preventing damage due to acute coronary syndromes by administering a pharmaceutical formulation comprising Apo A-I Milan. In a preferred embodiment, Apo A-I Milano can form a complex with lipids. In a more preferred embodiment, Apo A-I Milano forms a complex with POPC.

Apo A-I Milano or the lipid complex thereof may be administered in the form of a pharmaceutical formulation. Pharmaceutical formulations as described in the present invention include, for example, the addition of acceptable diluents, excipients, vehicles or carriers. As is known in the art, the addition of one or more diluents, excipients, vehicles or carriers makes the formulation suitable for administration to the subject and confers other desirable properties such as extended shelf life. .

Pharmaceutical formulations generally include a pharmaceutically acceptable carrier or vehicle. Many pharmaceutically acceptable carriers or vehicles can be used. In the examples described herein sucrose-mannitol is used. Physiological saline is often used as a pharmaceutical carrier or vehicle. Other suitable carriers or vehicles include glucose, trehalose, sucrose, sterile water, buffered water, 0.45% saline (semi-physiological saline), and 0.3% glycine, and additionally for increased stability, such as albumin. It may further comprise glycoprotein. These formulations can be sterilized by conventional and well known sterilization techniques. The resulting aqueous solution can be packaged for use or filtered and lyophilized (freeze-dried) under aseptic conditions. The lyophilized formulation can then be combined with sterile aqueous solution prior to administration.

Pharmaceutical formulations may also contain pH adjusting and buffering agents, tonicity adjusting agents, such as pharmaceutically acceptable excipients such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, and calcium chloride as needed under appropriate physiological conditions. Can be. Antibacterial agents such as phenol, benzalkonium chloride or benzetonium chloride may be added to maintain the sterility of the product, particularly pharmaceutical formulations intended for several-dose parenteral use. Suspending, stabilizing and / or dispersing agents may also be used in the formulations of the present invention.

The pharmaceutical formulation may comprise apolipoprotein (apo A-I Milano) in salt form. For example, because proteins can include acidic and / or basic ends and / or side chains, Apo A-I Milan can be present as a free acid or base in pharmaceutical formulations or as a pharmaceutically acceptable salt. Pharmaceutically acceptable salts include, for example, inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid, and the like; And apo AI Milano salts, including organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid, and the like. Suitable acids may be included which can form. Suitable bases capable of forming salts with Apo A-I Milan include, for example, inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide and the like; And mono-, di- and tri-alkyl amines (eg triethyl amine, diisopropyl amine, methyl amine, dimethyl amine and the like) and optionally substituted ethanolamines (eg ethanolamine, diethanolamine and the like) Organic bases such as) may be included.

Pharmaceutical formulations may have a variety of forms suitable for any route of administration, including but not limited to parenteral, enteral, topical or inhalation. Parenteral administration refers to any route of administration that does not cross the digestive tract, including but not limited to injectable administration (ie, intravenous, intramuscular, etc. as described herein). Intestinal administration refers to any route of administration using the digestive tract, oral or rectal, including but not limited to tablets, capsules, oral solutions, suspensions, sprays, etc., as described herein. For the purposes of the present application, enteral administration also represents the vaginal route of administration. Topical administration means all routes of administration through the skin, including but not limited to creams, ointments, gels and transdermal patches as described herein (see also Remington's Pharmaceutical Sciences). , 18 ^th Edition Gennaro et al . , eds. Mack Printing Company, Easton, Pennsylvania, 1990).

Parenteral pharmaceutical formulations of the invention can be injected into, for example, intravenous (intravenous), arterial (intraarterial), muscle (intramuscular), subcutaneous (subcutaneously or depot preparation), pericardial, or coronary arteries. It can be administered by. Pharmaceutical formulations for injection may be pharmaceutically suitable formulations for direct administration to the heart, pericardium or coronary artery. In a preferred embodiment, the pharmaceutical formulation is infused into the subject's peripheral blood vessels, for example in the arm or vestibule.

Pharmaceutical formulations for injection may be sterile suspensions, solutions or emulsions in aqueous or oily vehicles. Injectable formulations may, for example, be in unit dosage form in ampoules or in multidose containers, and may include added preservatives. Preferred buffers for parenteral pharmaceutical preparations are phosphate, citrate and acetate.

In another embodiment, the pharmaceutical formulation is in powder form for reconstitution with a suitable vehicle, including but not limited to sterile water, saline or dextrose that does not contain pyrogenic substances prior to use. Can be provided. To this end, Apo A-I Milano can be lyophilized as required or co-freeze dried with lipids. In another embodiment, the pharmaceutical formulation may be supplied in unit dosage form and re-formulated prior to use.

For long term delivery, pharmaceutical formulations may be provided in depot formulations for administration by implantation, for example by subcutaneous, intradermal or intramuscular injection. Thus, for example, pharmaceutical formulations may use suitable polymeric or hydrophobic materials (eg, as emulsions in acceptable oils) or ion exchange resins, or as poorly soluble derivatives, eg, Apo AI Milan or Apo AI It may be formulated in the form of a sparingly soluble salt of the Milano: lipid complex.

In another embodiment, the pharmaceutical formulation is a transdermal delivery system prepared as an adhesive disk or patch that slowly releases the active ingredient for transdermal absorption. In such embodiments, a permeation enhancer can be used to promote percutaneous penetration of Apo A-I Milan. In another embodiment, the percutaneous pharmaceutical formulation may further contain nitroglycerin for use in patients with angina.

For administration by inhalation, the pharmaceutical formulation is pressurized pack with a suitable propellant, for example dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. Can be delivered by nebulizer, or by spray from nebulizer. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve, for example a metered dose inhaler, to deliver a metered amount. Capsules and cartridges of gelatin can be formulated, for example, for use in an inhaler or insulator, containing a powder mixture of apo A-I Milano and lactose or starch.

The formulation may, if necessary, be present as a pack or dispenser device which may include one or more unit dosage forms containing the Apo A-I Milano pharmaceutical formulation. The pack may for example consist of a metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions or labeling for administration.

In certain embodiments, the pharmaceutical formulation of Apo AI-M or Apo A-I Milano: Repid complex may comprise a concentration of Apo A-I Milan sufficient to treat a subject in need of treatment. In certain embodiments, the pharmaceutical formulation of Apo A-I Milano or Apo A-I Milano: Lipid complex may comprise Apo A-I Milano at a concentration of about 5 mg / ml to about 50 mg / ml. In a preferred embodiment, the formulation may comprise Apo A-I Milano at a concentration of about 10 mg / ml to about 20 mg / ml. In a more preferred embodiment, the formulation may contain Apo A-I Milano at a concentration of about 13 mg / ml to about 16 mg / ml. Concentrations of Apo A-I Milan can be measured by any suitable technique known to those skilled in the art. In certain embodiments, the concentration of Apo A-I Milano is measured by size exclusion high performance liquid chromatography (SE-HPLC).

In certain embodiments, the pharmaceutical formulation of the Apo AI-M or Apo A-I Milano: Lipid complex may contain a concentration of lipids sufficient to form a complex with Apo A-I Milano. In certain embodiments, the pharmaceutical formulation of Apo A-I Milano or Apo A-I Milano: Lipid complex may contain POPC at a concentration of about 1 mg / ml to about 50 mg / ml. In a preferred embodiment, the formulation may contain POPC at a concentration of about 5 mg / ml to about 25 mg / ml. In a more preferred embodiment, the formulation may contain POPC at a concentration of about 10 mg / ml to about 20 mg / ml. In even more preferred embodiments, the formulation may contain POPC at a concentration of about 11 mg / ml to about 17 mg / ml. The concentration of POPC can be measured by any suitable technique known to those skilled in the art. In certain embodiments, the concentration of POPC is measured by high performance liquid chromatography.

In a preferred embodiment, the pharmaceutical formulation of the Apo A-I Milano: Lipid complex may contain sucrose in an amount sufficient to prepare a pharmaceutically suitable formulation of the Apo A-I Milano or Lipo Complex. In certain embodiments, the pharmaceutical formulation may contain about 0.5% to about 20% sucrose. In certain embodiments, the pharmaceutical formulation may contain about 3% to about 12% sucrose. In certain embodiments, the pharmaceutical formulation may contain about 5% to about 7% sucrose. In a preferred embodiment, the pharmaceutical formulation may contain about 6.0% to about 6.4% sucrose. In the most preferred embodiment, the pharmaceutical formulation may contain 6.2% sucrose.

In a preferred embodiment, the pharmaceutical formulation of the Apo A-I Milano: Lipid complex may contain mannitol in an amount sufficient to prepare a pharmaceutically suitable formulation of the Apo A-I Milano or Lipo Complex. In certain embodiments, the pharmaceutical formulation may contain about 0.01% to about 5% mannitol. In certain embodiments, the pharmaceutical formulation may contain about 0.1% to about 3% mannitol. In certain embodiments, the pharmaceutical formulation may contain about 0.5% to about 2% mannitol. In a preferred embodiment, the pharmaceutical formulation may contain about 0.8% to about 1% mannitol. In the most preferred embodiment, the pharmaceutical formulation may contain 0.9% mannitol.

In certain embodiments, the pharmaceutical formulation of the Apo A-I Milano: Lipid complex may contain a buffer in an amount sufficient to prepare a pharmaceutically suitable formulation of the Apo A-I Milano or Lipo Complex. In certain embodiments, the pharmaceutical formulation may comprise a phosphate buffer. In certain embodiments, the buffer concentration may be about 3 mM to about 25 mM. In certain embodiments, the buffer concentration may be about 5 mM to about 20 mM. In a preferred embodiment, the buffer concentration may be about 8 mM to about 15 mM. In certain embodiments, the pH of the pharmaceutical formulation is adjusted to a range suitable for administration to the subject by the addition of appropriate buffers. In certain embodiments, the pharmaceutical formulation may have a pH of about 6.8 to about 7.8. In certain embodiments, the pharmaceutical formulation may have a pH of about 7.0 to about 7.8. In certain embodiments, the pharmaceutical formulation may have a pH of about 7.2 to about 7.5. In a preferred embodiment, the pharmaceutical formulation may have a pH of about 7.5.

In certain embodiments, the pharmaceutical formulation of Apo A-I Milano: Repid complex has an osmolality suitable for administration to a subject. In certain embodiments, the osmolality of the formulation can be about 200 to about 400 mOsm. In certain embodiments, the pharmaceutical formulation may have an osmolality of about 220 to about 380 mOsm. In certain embodiments, the pharmaceutical formulation may have an osmolality of about 260 mOsm to about 340 mOsm. In a preferred embodiment, the pharmaceutical formulation may have an osmolality of about 280 mOsm to about 320 mOsm. In the most preferred embodiment, the pharmaceutical formulation may have an osmolality of about 290 mOsm.

The formulations of the present invention provide apo A-I Milano: Lipid complex of sufficient purity to be administered to a subject. In certain embodiments, the pharmaceutical formulation is about 98% or more, about 96% or more, about 95% or more, about 93% or more, about 91% or more or about 90% or more It may contain Apo AI Milan in purity. In a preferred embodiment, the purity of Apo A-I Milano is about 90% or more. The purity of Apo A-I Milano can be measured by any suitable technique known to those skilled in the art. In certain embodiments, the purity of Apo A-I Milano can be measured by size exclusion HPLC.

The formulations of the present invention provide apo A-I Milano: Lipid complex of POPC of sufficient purity to be administered to a subject. In certain embodiments, the pharmaceutical formulation is about 98% or more, about 96% or more, about 95% or more, about 93% or more, about 91% or more or about 90% or more It may contain POPC in purity. In a preferred embodiment, the purity of the POPC is greater than about 90%. The purity of the POPC can be measured by any suitable technique known to those skilled in the art. In certain embodiments, the purity of the POPC can be measured by HPLC.

In certain preferred embodiments, the Apo AI Milano: Lipid complex is about 10% or less, about 8% or less, about 6% or less, about 4% or less, about 2% or less, about 1% or iron (II) oxidation / xyleneol orange assay (Jiang, et al . 1992, Anal . Biochem 202: 384-389) and the amount of lipid hydroperoxide below the detectable limit as measured by Biochem 202: 384-389). In certain embodiments, the Apo AI Milano: Lipid complex has a purity (measured in% of total peak area) of greater than 85% as measured by gel permeation chromatography. In certain embodiments, the formulation contains little or no endotoxin. In a preferred embodiment, the formulation has an endotoxin of <0.04 EU per mg of Apo AI Milan.

In certain embodiments, the formulation may be <about 6,000 per 50 ml vial as the amount of microparticles greater than 10 μm in size measured by light obscuration. In certain embodiments, the amount of particulate greater than 25 μm in size is <about 600 per 50 ml vial as measured by light attenuation.

In a preferred embodiment, Apo A-I Milano: Lipid Complex Pharmaceutical Formulation is prepared by diluting recombinant Apo A-I Milano with a water for injection to a concentration of 15 mg / ml in solution. Sodium phosphate is added to a final concentration of 9-15 mM and the pH is adjusted between about 7.0 and about 7.8. Mannitol is added to obtain a concentration of about 0.8% to about 1% mannitol (w / v). POPC is then added to obtain a ratio of Apo A-I Milano to POPC of 1: 0.95 (wt protein / wt lipid). The mixture is stirred at 5000 rpm for about 20 minutes using an overhead propeller and Ultra Turax while maintaining the temperature between 37 ° C. and 43 ° C. The temperature is maintained between 32 ° C. and 43 ° C. using an in-line heat exchanger (Avestin, Inc.) while the feed vessel is continuously stirred at 300 rpm. Homogenization for the first 30 minutes is carried out at 50 MPa (7250 psi), after which the pressure is 80- until the test during preparation by gel permeation chromatography shows AUC% greater than about 70% between protein standards. Maintain at 120 MPa (11600-17400 psi). Thereafter, the osmolality of the composite is adjusted by adding 6.0% to 6.4% sucrose. The Apo A-I Milano: POPC complex is then sterilized by filtration through a 0.22 μM filter.

In another preferred embodiment, the pharmaceutical formulation has an osmolality of about 280 mOsm to about 320 mOsm and contains about 12 to about 18 mg / ml of recombinant Apo AI Milan, pH 7.4, about 11 to about 17 mg / ml. 6.2% sucrose and 0.9% mannitol together with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. The pharmaceutical formulation may have rApo A-I-M of about 90% purity and POPC of about 97% purity. In certain embodiments, no single impurity can be greater than about 2%.

Pharmaceutical formulations can be frozen and stored (about -15 ° C to about -25 ° C). In certain embodiments, the formulation may be a cold solution, frozen solution or lyophilized solution. Such agents are preferably thawed and warmed to room temperature prior to administration to the subject. Gentle thawing and warming are recommended to avoid protein denaturation.

In another preferred embodiment, the formulation is about 2 ml to about 250 ml, preferably about 10 ml to about 100 milliliters, most preferably about 50, containing a pharmaceutical formulation comprising the Apo AI Milano: phospholipid complex May be present in ml sterile glass vials. In certain embodiments, the pharmaceutical formulation may comprise from about 10 mg / ml to about 15 mg / ml of the Apo A-I Milano: Phosporipid complex at a final fill dose of about 39-41 ml per vial. The amount of apo A-I Milano: phospholipid complex may be from about 500 mg to 750 mg per 50 ml vial.

The pharmaceutical formulation may be for a single single use or may contain an antimicrobial excipient as described above to provide a pharmaceutical formulation suitable for multiple use, eg, a multiple-use vial. In certain embodiments, the pharmaceutical formulation may be in a unit-of-use package. As is known to those skilled in the art, the use-unit package is a convenient prescription sized patient ready-to-use unit labeled for direct distribution by a health care provider. The use-unit package contains the pharmaceutical formulation in the amount required for a representative treatment interval and duration for a given indication. The methods and formulations of the present invention comprise a pharmaceutical formulation comprising the Apo AI Milano: phospholipid complex in an amount sufficient to, for example, treat an average sized adult male or female at 15 mg / kg once a week for 5 weeks. It is available in a use-unit package. In one embodiment, the use-unit package may contain a pharmaceutical formulation comprising the Apo AI Milano: phospholipid complex in an amount sufficient to treat an average sized adult subject at 45 mg / kg once weekly for 6 weeks. have. It will be apparent to those skilled in the art that the doses described herein are based on the weight of the subject.

Pharmaceutical formulations include, but are not limited to, the formulations contained, and instructions for use, dosages, dosage intervals, duration, indications, contraindications, warnings, precautions, handling and storage instructions, cardiovascular and blood vessels Labels may be attached or have attached labels to identify palliative care providers and other information useful for the treatment and prevention of diseases, acute coronary syndromes, ischemic diseases, and stabilization of plaques.

In a further embodiment, the present invention provides a kit for treating or preventing cardiovascular and vascular diseases, acute coronary syndromes, ischemic diseases and stabilizing plaques. The kit is used together with one or more effective amounts of Apo AI Milano or Apo AI Milano: Lipid complex or a pharmaceutical formulation thereof to treat or prevent Apo AI Milano or Apo AI Milano: Lipid according to the ice method of the present invention. Labels or labeling with instructions for using the complex or pharmaceutical formulations thereof. In certain embodiments, the kit is a component useful for carrying out this method, such as an Apo AI Milano or an Apo AI Milano: Lipid complex or a device for delivering a pharmaceutical formulation thereof, and a component, eg, for the safe processing of these devices. For example, it may include a sharps container. In certain embodiments, the kits may comprise Apo A-I Milano or Apo A-I Milano: Lipid complex or pharmaceutical formulation thereof in a pre-filled syringe, unit-dose or use-unit package.

The invention will be better understood with reference to the following non-limiting examples.

Example  One: ETC -216 tolerance

This example demonstrates the safety and tolerability of ETC-216 in healthy volunteers.

Double-blind placebo-controlled trials were performed to determine the safety and tolerability of Apo A-I Milano and phospholipids. Apo A-I Milano forms a complex in a 1: 1 weight ratio with phospholipid (POC) and is called ETC-216. This trial evaluates the safety and tolerability of five incremental doses of one intravenous infusion of ETC-216 in healthy male volunteers and two different doses in healthy female volunteers. Informed consent was obtained from all volunteers prior to the test.

Intravenous doses of ETC-216 were administered to 32 healthy volunteers (“subjects”) aged 18-50 years old. Male subjects were administered a dose of up to 100 mg / kg. Female subjects were administered a dose of up to 50 mg / kg. The sex, dose and rate of the subjects are presented in Table 1 below.

Dose (mg protein / kg body weight) Number of objects gender Injection rate (mg / kg / min) 0 (placebo) 3 male 1.67 0 (placebo) 2 male 1.25 5 3 male 1.67 15 3 male 1.67 50 3 male 1.67 75 3 male 1.25 100 3 male 1.25 0 (placebo) One female 1.67 0 (placebo) 2 female 0.83 15 3 female 1.67 15 3 female 0.83 50 3 female 0.83

Adverse event monitoring includes clinical laboratory evaluations, physical examinations, and vital signs. Subjects were monitored for 27 days after dosing.

In this study, 20 of the 32 subjects reported adverse events, all of which were mild to moderate. Adverse events in two or more subjects were thought to be related to the treatment of the test drug. These side effects and the number of subjects who experienced these side effects were lymphopenia (11 subjects), leukocytosis (10 subjects), nausea (6 subjects), headache and diarrhea (4 subjects), vomiting (3 subjects) And abdominal pain and hypertriglyceridemia (2 subjects). Most of the relevant side effects (gastrointestinal and leukocyte disease) have been reported after injecting 50 mg / kg or 100 mg / kg of ETC-216 into a male subject. Two side effects (hypoproteinemia and abnormal liver function) seen in placebo-treated subjects were thought to be related to the trial treatment. There were no deaths, serious side effects, or discontinuation due to side effects.

As shown in FIG. 1, 30 minutes after administration of a single dose of ETC-216, serum levels of HDL non-esterified cholesterol in male subjects increased at doses of 15 mg / kg and above.

Antibody titers against Apo A-I Milano were tested in serum prior to and 27 days post infusion. There was no significant antibody response in any subject after single dose administration.

The terminal half-life of about 100 hours supports the rationale for performing initial multiple dose tests in humans using the dosing regimen every 7 days.

These results suggest that ETC-216 was safe and well tolerated at all doses, with no serious side effects.

Example  2: degenerative atherosclerosis in  Of various capacities ETC -16 Benefits

This example demonstrates the efficacy of the 15 mg / kg and 45 mg / kg doses of ETC-216 on degenerative coronary atherosclerosis in subjects with acute coronary syndrome.

This test, as assessed by intravascular ultrasound, was a randomized, double-blind placebo-controlled, multiple-dose trial to evaluate the efficacy and safety of ETC-216 in subjects with acute coronary syndromes. Prior to initiating any test-specific procedure, all subjects received informed consent approved by their Institutional Review Board (IRB).

Eligible subjects are diagnosed with acute coronary syndrome (unstable angina, non-Q wave myocardial infarction or ST elevation myocardial infarction) within 14 days prior to screening, perform coronary angiography and / or percutaneous coronary intervention (PCI) The subject was scheduled to maintain the expected 24-hour following this procedure. Subjects meeting these criteria were excluded if their procedures were considered to be emergency and / or as determined by the practitioner and were accompanied by a high risk of acute complications.

The primary efficacy endpoint is the change in percent atherosclerosis volume at 30-80 mm sections of one targeted (imaged) coronary artery as assessed by intravascular ultrasound (IVUS) (at end of treatment-pretreatment). ). Positive results were defined as the negative change in percent atheros vol- ume with a confidence interval (CI) not containing zero.

Secondary efficacy endpoints were mean change in total atherosclerosis volume and mean maximum atherosclerosis thickness. Secondary efficacy endpoints were set for IVUS and angiography. For IVUS, the secondary efficacy endpoints were as follows:

1) nominal change in total plaque “volume” as measured by the mean of all plaque areas for all slices of anatomically equivalent sections of the target coronary artery (end of treatment—pretreatment);

2) nominal change in mean maximum plaque thickness for all slices of anatomically equivalent sections of the target coronary artery (end of treatment—pretreatment);

3) minimum and maximum diseased sections "volume";

a) nominal change in section plaque “volume” for adjacent slices of 10 mm at baseline containing the minimum percent plaque area (defined as the least diseased section) (at the end of treatment—pretreatment);

b) Nominal change in section plaque “volume” for adjacent slices of 10 mm at baseline containing the maximum percent plaque area (defined as the largest diseased section) (at the end of treatment—pretreatment).

Secondary efficacy endpoints for angiography were:

1) mean coronary lumen diameter in all measured coronary sections;

2) number of lesions of new angiograms in each treatment subgroup;

3) the proportion of subjects with one or more new coronary lesions at the end of treatment;

4) number of sites with existing lesions indicating degeneration;

5) number of sites with existing lesions showing progression;

6) the proportion of subjects showing "regression" in any existing lesion;

7) the proportion of subjects showing “progressive or invariant” in all existing lesions;

8) Percentage of subjects showing "regression or invariance" in all existing lesions.

The method for IVUS was analyzed via this name as described in Nissen 2001, Am . J. Cardiol . 87: 15A-20A, which is incorporated herein in its entirety. Briefly, the operator digitizes the IVUS videotape, examines the pullback, and selects the most distal side-branch as a starting point for analysis as shown in FIG. 2 (A). The origin was chosen. Subsequently, every 30th image was selected for analysis, which represents a series of cross sections spaced exactly 0.5 mm apart. The final analyzed cross section was the proximal image in sequence before the appearance of the left main coronary artery or the right coronary artery (proximal fiduciary site), as shown in FIGS. 2 (B) -2 (D). This method was repeated for follow-up using the same landmark to ensure that the same section was analyzed at both time points.

Both directed and induced IVUS measurements were performed. Direct IVUS measurements were performed according to the criteria of the American College of Cardiology and the European Society of Cardiology (Mintz et al. al . 2001, J. Am. Coll . Cardiol. 37: 1478-92; This full name is incorporated herein in its entirety). Using the National Institute of Health Image 1.62 (NIH public domain software), the operator measures the 1-mm grid marks encoded by the scanner into IVUS images. (calibration procedure) was performed. For each cross section, the operator performed a manual area measurement to track the leading egde of the lumen and outer elastic membrane border (FIG. 3 (A) -mm 2 3 (D)). In addition, the maximum atherosclerosis thickness was measured directly. The accuracy and reproducibility of these methods have already been reported, which indicates that after testing the average IVUS cross-sectional measurements are true dimensions for precision-drilled Lucite phantoms ranging from 3.24 mm 2 to 27.99 mm 2 area. Proved to be within 0.5% (Schoenhagen et al . 2003, J. Am . Soc . Echocardiogr. 16: 277-84; This full name is incorporated herein in its entirety). The variability of measurements by multiple observers showed a standard deviation of 2.9%.

Derived IVUS measurements included the calculation of the atheroscopy area as the lumen area minus the outer elastic membrane (EEM) area. Since image cross sections were obtained at 0.5-mm intervals, the total atheroscopy volume could be calculated by multiplying the average atheros area by the pullback length in mm using the Simpson rule. The percent atheros volume was calculated as follows: (Atherom area / EEM area) × 100 (Reference: Nissen et al ., 2003, JAMA 290: 2292-2300). Analysis of coronary angiography was performed in the core laboratory of the Cleveland Clinic Foundation using standardized methods designed to reduce measurement variability. The system was assayed by comparing the diameter of the angiographic catheter tip to its known dimensions. Angiographic endpoints were changes in mean coronary lumen diameter from baseline to follow-up.

57 subjects were randomly administered every 7 days with 15 mg / kg ETC-216, 45 μg / kg ETC-216 or equivalent volume placebo at an intravenous dose of up to 5. Subjects were randomized at a ratio of 2: 2: 1 for 15 mg / kg ETC-216, 45 mg / kg ETC-216 or placebo. 47 subjects completed the test.

The 15 mg / kg dose was injected intravenously over about 50 minutes and the 45 mg / kg dose was injected intravenously over about 150 minutes. The infusion rate was increased for some subjects due to the onset of nausea. All intravenous infusions were done peripherally.

Subjects were monitored by clinical laboratory parameters, including antibody level, physical examination, vital signs, electrocardiogram (EKG), and frequency and intensity of clinical side effects to ETC-216. Angiography and IVUS were performed twice for each patient. The first period was after the completion of the initial screening visit, and the second was about 2 weeks (± 7 days) after the last dose of the test drug.

Positive results were obtained with respect to the primary endpoint as summarized in Table 2 below. In the combination treatment group (patients administered the 15 mg / kg or 45 mg / kg dose of ETC-216), the change in the mean (standard deviation or SD) percent of atherosclerosis was -1.06% (3.17%). The median was -0.81% (95% CI, -1.53% to -0.34%; p = 0.02 compared to baseline). In the placebo group, the mean (SD) change was 0.14% (3.09%). The median was 0.03% (95% CI, -1.11% to 1.43%; p-0.97 compared to baseline).

Atherom volume percentage in target coronary artery slice base line Tracking Change from baseline Treatment group Number of objects Average (SD ¹ ) Median (IQR ² ) Mean (SD) Median (IQR) Mean (SD) Median (95% CI ³ ) P value ⁴ Placebo 11 34.80 (8.44) 35.29 (26.99- 39.81) 34.94 (9.74) 33.59 (27.07- 41.09) 0.14 (3.09) 0.03 (-1.11-1.43) .97 ETC-216 15 mg / kg 21 39.71 (7.04) 40.55 (34.16- 44.97) 38.42 (6.5) 38.84 (32.78- 42.87) -1.29 (3.48) -1.14 (-2.24- -0.56) .03 ETC-216 45 mg / kg 15 37.92 (7.83) 41.22 (31.92- 44.10) 37.19 (7.71) 36.62 (32.81- 44.02) -0.73 (2.75) -0.34 (-01.21- 0.43) .45 ETC-216 combination 36 38.96 (7.32) 40.88 (34.01- 44.53) 37.91 (6.95) 38.17 (32.79- 43.53) -1.06 (3.17) -0.81 (-1.53- -0.34) .02 ^{5 1} standard deviation ² quartile range ³ confidence interval ⁴ P value for intragroup comparison from Wilcoxon signed rank test. For group-to-group comparison of ETC-216, Apo AI Milano: POPC combination versus placebo, from analysis of covariance of the grade of change from baseline with baseline values as covariates, P = 0.29. ⁵ pre-specified primary efficacy endpoints

Regarding secondary endpoints, positive results were also obtained, as shown in Table 3 below. Compared to baseline, the mean (SD) change in total atherosclerosis in the combination treatment group was -14.1 mm 3 (39.5 mm; median -13.3 mm 3; 95% CI, -20.7 to -7.2; p <0.001). For the placebo group, the corresponding change was -2.9 ㎣ (23.3 ㎣). Median was -2.9 mm 3 (95% CI, -8.6 to 8.2; p = 0.97). The mean (SD) change from baseline in maximum atherosclerosis for the combination treatment group was -0.042 mm (0.080 mm). Median was -0.035 mm (95% CI, -0.058 to -0.020; p <0.002). For the placebo group, the corresponding change was -0.008 mm (0.061 mm; median -0.009 (95% CI, -0.035 to 0.026; p = 0.83). See Table 4 below.

Total Atherom Volume in the Target Coronary Artery Section Baseline, ㎣ Tracking Change from baseline, ㎣ Treatment group Number of objects Mean (SD) Median (IQR) Mean (SD) Median (IQR) Mean (SD) Median (95% CI) P value ¹ Placebo 11 172.6 (113.2) 149.0 (58.6- 216.5) 169.8 (118.0) 145.1 (58.4- 217.8) -2.9 (23.3) -0.2 (-8.6- 8.2) .97 ETC-216 15 mg / kg 21 295.5 (166.5) 279.7 (173.6- 370.7) 280.4 (155.6) 265.5 (161.1- 351.6) -15.1 (50.6) -15.0 (-29.6- -4.9) .02 ETC-216 45 mg / kg 15 230.6 (156.7) 195.8 (90.9- 332.9) 218.0 (145.6) 183.8 (89.4- 311.1) -12.6 (15.3) -12.0 (-20.6- -2.9) .007 ETC-216 combination 36 268.4 (163.5) 263.7 (129.4- 359.3) 254.4 (152.6) 244.8 (131.0- 337.6) -14.1 (39.5) -13.3 (-20.7- -7.2) <0.001 ^{2 1} P value for intragroup comparison from Wilcoxon signed rank test. For group-to-group comparison of ETC-216, Apo AI Milano: POPC combination versus placebo, from an analysis of the covariance of the grade of change from baseline with baseline values as covariates, P = 0.18. ² pre-specified primary efficacy endpoints

Average maximum atherosclerosis thickness in the target coronary artery section Baseline, mm Tracking, mm Change from baseline, mm Treatment group Number of objects Mean (SD) Median (IQR) Mean (SD) Median (IQR) Mean (SD) Median (95% CI) P value ¹ Placebo 11 0.649 (0.317) 0.683 (0.402- 0.846) 0.641 (0.332) 0.584 (0.392- 0.827) -0.008 (0.061) -0.009 (-0.035- 0.026) .83 ETC-216 15 mg / kg 21 0.815 (0.194) 0.830 (0.683- 0.953) 0.771 (0.168) 0.824 (0.646- 0.865) -0.044 (0.094) -0.049 (-0.079- -0.015) .03 ETC-216 45 mg / kg 15 0.738 (0.280) 0.792 (0.531- 0.992) 0.699 (0.269) 0.758 (0.457- 0.963) -0.039 (0.059) -0.029 (-0.056- -0.007) .02 ETC-216 combination 36 0.783 (0.233) 0.816 (0.578- 0.963) 0.741 (0.216) 0.814 (0.552- 0.895) -0.042 (0.080) -0.035 (-0.058- -0.020) <0.001 ^{2 1} P value for intragroup comparison from Wilcoxon signed rank test. For group-to-group comparison of ETC-216, Apo AI Milano: POPC combination versus placebo, from an analysis of the covariance of the grade of change from baseline with baseline values as covariates, P = 0.23. ² pre-specified primary efficacy endpoints

The results showed that ETC-216 was effective in reducing plaque size. Statistically significant degeneration of atherosclerosis was demonstrated in both treatment groups.

Example  3: In vitro Langendorf  ( Ex vivo Langendorff )

This example demonstrates the cardioprotective effect of prophylactic ETC-216 in reperfusion isolated ischemic rabbit heart. Male New Zealand white rabbits weighing about 2-3 kg, obtained from Charles River, were used in this test. Male New Zealand white rabbits were selected as the appropriate test system for the purposes of this test. Isolated ischemic-reperfusion rabbit heart is a model of human myocardial infarction. Upon arrival the animal assigned a unique identification number.

Animals were housed in stainless steel cages according to the guidelines of the University of Michigan Committee on the Use and Care of Animals. Veterinary care was provided by the University of Michigan Unit for the Laboratory Animal Medicine. The University of Michigan has been approved by the American Association of Accreditation of Laboratory Animal Health Care, and the Animal Care Use Program is a standard in the Guide for the Care and Use of Laboratory Animals (DHEW (NIH) Publ. No. 86-23).

ETC-216 is a recombinant apolipoprotein A-I Milan / 1-palmitoyl-2-oleoyl phosphatidylcholine complex in a 1: 1 weight ratio. The stock of ETC-216 contained 14 mg protein / ml in sucrose mannitol buffer. Since sucrose-mannitol buffer is incompatible with Krbs-Henseleit buffer, and in order to control any independent effects with mannitol alone, it is dialyzed with ETC-216 in 4 mM sodium phosphate. Background buffer consisting of 2% glucose (pH 7.4) was obtained. ETC-216 was diluted with Krebs-henselite buffer to obtain a drug concentration of 0.45 mg / ml. Vehicles were similarly diluted.

Dose selection was based on historical data on doses used in Esperion's Human Phase I single dose safety clinical trials, where humans were dosed with ETC-216 at doses up to 100 mg / kg. . For the tests outlined in this protocol, the concentration of 0.5 mg / ml corresponds approximately to the intravenous dose of 25 mg / kg administered to humans.

Experiments were performed using a Langendorf apparatus to perfuse rabbit hearts. Rabbits were dislocated cervically to become unconscious, and the heart was quickly removed and attached to the cannula for perfusion through the aorta. The perfusion medium is continuously exposed to a mixture of 95% O ₂ /5% CO ₂ and with circulating Krebs-Hesselite buffer (pH 7.4, 37 ° C .; “KH”) delivered at a constant rate of 20-25 ml / min. Configured. The heart was tuned through an electrode attached to the right atrium throughout the protocol. Tunable stimuli were delivered from a laboratory square wave generator (at least 10% physiological tuning, 1 msec period, Grass 588, Quincy, MA). The pulmonary artery was inserted by cannula by SilasticTM tubing to facilitate the collection of pulmonary artery effluents indicating coronary venous return to the coronary sinus. Relative and inferior vena cava and pulmonary veins were ligated to prevent loss of perfusion fluid from other vessels. The left ventricular drain tube, thermistor probe, and latex balloon were inserted through the left atrium and placed in the left ventricle. The fluid-filled latex balloons were connected to Miller Catheter pressure transducers by rigid tubing to allow measurement of left ventricular pressure. The intraventricular balloon was inflated with distilled water to obtain an initial baseline left ventricular dilatation pressure of about 10 mmHg. Coronary perfusion pressure was measured with a pressure transducer connected to the side-arm of the aortic cannula. Since the rate of coronary perfusion remained constant, the change in coronary perfusion pressure served as an indicator of the change in coronary resistance. All hemodynamic parameters were continuously monitored using a multichannel recorder such as Grass Polygraph 79D (Quincy, Mass.) Connected to a Polyview Software Data Acquisition System. The heart was maintained at 37 ° C. throughout the experiment by enclosing the heart in a temperature controlled dual lumen glass chamber and passing the perfusion medium through a heated reservoir and delivery system.

Two treatment groups were used for the experimental procedure as shown below.

group cure Test substance Concentration (mg / ml) One Ischemia and Reperfusion Vehicle 0 2 Ischemia and Reperfusion ETC-216 0.45

The heart was treated experimentally as shown in FIG. 5. Isolated heart was stabilized under normoxic (normal levels of oxygen) for 20 minutes before inducing total ischemia. During the first 10 minutes of this period, the heart was exposed only to KH buffer and then for an additional 10 minutes to KH buffer containing vehicle (group 1) or ETC-216 (group 2). The heart was then subjected to a 30 minute ischemic period followed by a 60 minute reperfusion period with KH buffer containing vehicle (group 1) or ETC-216 (group 2). Induction of total total ischemia was achieved by stopping the flow of perfusion to the heart, and reperfusion of the heart was done by running the pump to restore the original flow rate.

An aliquot of the pulmonary artery effluent was collected from the heart of all groups at baseline (pre-ischemic), and primarily every 5 minutes up to 5 minutes and then every 5 minutes during the reperfusion period. The effluent was analyzed for creatine kinase concentration (FIG. 6) and tissue damage index. Creatine kinase is a cytosol enzyme released from irreversibly damaged cells. Cardiac function was continuously monitored (FIG. 7).

Cardiac endpoint measurements were made for:

ECG-heart rate (tuned) to detect for the presence or absence of 1-arrhythmia;

2-left ventricular incidence pressure (FIG. 8) (data shown as standard error of mean ± mean for a specified number of hearts in each group);

3-left ventricle dP / dt

4-left ventricular dilatation pressure (FIG. 9) (data shown as standard error of mean ± mean for a specified number of hearts in each group);

5-coronary perfusion pressure (FIG. 10) (data shown as standard error of mean ± mean for a specified number of hearts in each group);

Collection of Lymphatic Drainage to Measure Tissue Creatine Kinase Release Before and After 6-Reperfusion (FIG. 6).

At the end of the experimental protocol, cardiac tissue biopsies obtained from up to 5 hearts from each treatment group were immediately immersed in liquid nitrogen and stored at −80 ° C. for subsequent liquid hydroperoxide analysis. Homogenate samples were normalized to protein content prior to analysis of lipid peroxides (FIG. 11). ETC-216 reduced cardiac lipid hydroperoxide by 46% in this example.

Upon completion of the designated protocol, two hearts from each group were perfused with 2.5% glutaraldehyde and 1% LaCl ₃ in 0.1 M sodium caltodylate buffer (pH 7.4) for 3 minutes. Under normal conditions, high osmotic affinity LaCl ₃ is maintained in the vascular ruptures bound to the vessel wall and serves as an indicator of vascular integrity. The outflow of LaCl ₃ into the extravascular space was used to indicate the presence of vascular damage. Tissue samples were separated from the left ventricular myocardial layer and cut into sections about 1 mm in size on one side. Samples were fixed for an additional 2 hours at 4 ° C. in the buffer mentioned above. Thereafter, samples were dehydrated in ethanol series and embedded in EM bed-812 (Electron Microscopy Sciences, Ft, Washington, PA). Tissue blocks were cut with Reichert ultramicrotome, placed on a formvar-coated copper grid, and then stained with 4% uranil acetate. Cross sections were observed with a Phillips CM-10 electron microscope.

Myocardial specimens from each of the test groups were examined using transmission electron microscopy. The image showed that the eradic structural features of the vehicle-treated heart were lost, and there was a contractor band. Mitochondria are markedly swollen by disintegrating crystals and high osmotic affinity inclusion bodies. In the ETC-216 treated heart, the eradication structure is relatively normal and the mitochondria are intact with only minimal swelling. The substantial absence of the constructive bands is in sharp contrast to that observed in the control heart. The ability of ETC-216 to prevent constructive band necrosis is consistent with the observation that the heart pretreated by ETC-216 did not show an increase in LVEDP upon reperfusion. Both construction band necrosis and continuous increase in LVEDP are associated with intracellular calcium overload and irreversible cellular damage. The presence of myofibrillar contamination of the Z-band, and disruption of the myofibrillar structure, is indicative of extensive damage. Other anticipated morphological damages include the collapsed crista and matrix of the mitochondria and the large electron dense bodies in the mitochondria. Magnification was 7900 × in both micrographs (FIG. 12).

Analysis of creatine kinase concentration (FIG. 6) suggested that the high speed phase of enzyme release into the venous effluent appeared at the time of reperfusion. Control heart (treated with vehicle) showed significant release of creatine kinase compared to ETC-216 treated heart. In addition, the ETC-216 treated heart had reduced left ventricular dilatation pressure (FIGS. 7 and 9), elevated left ventricular incidence pressure (FIG. 8), decreased coronary perfusion pressure (FIG. 10) and decreased liquid hydroperoxide ( LHP) (FIG. 12). In addition, ETC-216 protected against morphological changes in the myocardial layer (FIG. 12). These results demonstrate the cardioprotective effect of ETC-216 when administered prior to an ischemic attack.

Example  4: LAD  occlusion- Reperfused  Acute and Chronic Administration at 100 mg / kg in Rabbit Heart

This example demonstrates the cardioprotective effect of ETC-216 in an in vivo model of focal myocardial ischemia and reperfusion. This was used as a suitable test system because male New Zealand white rabbits lack a lateral blood supply to the heart, making it unnecessary to use myocardial blood flow measurements. In this trial, different dosage regimens were used in different groups of rabbits applied to 30 minutes of local myocardial ischemia by coronary ligation and reperfusion. Two dosage regimens were used. In the first protocol, ETC-216 was tested in a single pretreatment, where the heart was exposed to 100 mg / kg of drug immediately before the onset of focal ischemia, while in the second protocol one day before and immediately before the onset of focal ischemia Two 100 mg / kg pretreatments were administered. These protocols are shown in FIG. This study focused on the effect of ETC-216 as a cardioprotectant in an in vivo test in which rabbit hearts were applied to focal myocardial ischemia for a period of 30 minutes and then to reperfusion for at least 4 hours. This example demonstrates that ETC-216 is a cardioprotective agent when administered after an ischemic attack.

The method used in this study is in accordance with the guidelines of the University of Michigan Committee on the Use and Care of Animals. Veterinary care was provided by the University of Michigan Unit for the Laboratory Animal Medicine. The University of Michigan has been approved by the American Association of Accreditation of Laboratory Animal Health Care, and the Animal Care Use Program is a standard in the Guide for the Care and Use of Laboratory Animals (DHEW (NIH) Publ. No. 86-23).

Male New Zealand white rabbits weighing about 2-3 kg, obtained from Charles River, were used in this test. Upon arrival the animal assigned a unique identification number. Rabbits were anesthetized by intramuscular administration of a mixture of xylazine (3.0 mg / kg) and ketamine (35 mg / kg) followed by intravenous injection of sodium pentobarbital solution (30 mg / ml). Anesthesia was maintained by intravenous injection of pentobarbital solution (30 mg / ml). A cuffed endotracheal tube was inserted and the animal was kept in positive pressure ventilation with room air. The right jugular vein was isolated and a cannula was inserted to administer ETC-216 or equivalent dose of vehicle. MillarTM catheter micro-tip pressure transducer placed directly on the aortic plate to isolate the right carotid artery, monitor the aortic blood pressure and obtain the first derivative (dP / dt) of the pressure pulse. Equipment was installed. Lead II electrocardiograms were monitored throughout the experiment. Left thoracotomy and pericardiotomy were performed, followed by left anterior descending (LAD) coronary artery. Silk sutures (3.0; Deknatel, Fall River, Mass.) Were passed through the back of the artery and both ends of the sutures were inserted into short polyethylene tubing. Downward pressure on the polyethylene tube, exerting upward tension on the free end of the suture, compresses the original coronary artery, causing vascular occlusion and local myocardial ischemia. The occlusion was maintained for 30 minutes, after which the tension on the sutures was released and the polyethylene tubing was released to allow reperfusion to occur. Focal myocardial ischemia was evidenced by the presence of sites in the distribution region of occluded blood vessels and by changes in electrocardiogram (ST-section elevation) consistent with the presence of cervical focal myocardial ischemia.

Key endpoint measurements consisted of measuring infarct size as a percentage of the left ventricle and as a percentage of the area at risk (FIGS. 14 and 15). At the end of the trial, heparin (1,000 U, intravenous) was administered to the anesthetized rabbits, after which they were euthanized. After the heart was excised, it was prepared for reperfusion with Krebs-Hencelite buffer at a constant flow rate of 22-24 ml / min through the aorta on the Langendorf apparatus. The heart was washed with buffer for 10 minutes to keep tissues clean. A 1% solution of triphenyltetrazolium chloride (TTC) in 45 mL phosphate buffer (pH 7.4) was perfused through the heart. TTC suggests the presence of formazan precipitate resulting from the reduction of TTC by coenzymes present in viable myocardial tissue by dividing the non-infarct myocardial layer into red brick color in the area of risk. Irreversible glandular tissue lacking cytosolic dehydrogenase cannot form formazan precipitate and appears pale yellow. The left anterior descending (LAD) artery was ligated at the same location as the area ligated during the induction of focal myocardial ischemia. The perfusion pump was stopped and 2 ml of Evans Blue 0.25% solution was slowly injected through the side-arm port connected to the aortic cannula. The dye was passed through the heart for 10 seconds to ensure a uniform distribution of the dye. The presence of Evans blue was used to distinguish left ventricular tissue that did not cause local ischemia as opposed to the risk site. The heart was separated from the perfusion device and cut in cross section obliquely to the right with respect to the vertical axis. The right ventricle, apex, and arterial tissue were discarded. Both surfaces of each cross section were traced on a clean acetate sheet. Copied and enlarged the image. Copies were scanned and downloaded to Adobe PhotoShop (Adobe Systems Inc., Seattle, WA). Normal left ventricular (NLV) non-hazardous areas, areas of risk and infarction are measured by counting the number of pixels occupying each area using Adobe Photoshop software. The total risk area is expressed as a percentage of the left ventricle. Infarct size is expressed as a percentage of risk area (ARR) (FIGS. 14 and 15).

Percent infarct of risk area, Percent infarct of left ventricle, and left ventricle in rabbits treated once (ie acute) or twice (ie chronically) with ETC-216 (100 mg / kg) or equivalent volume of vehicle Percent of danger area is shown. Data is standard error of mean ± mean for n = 6 animals per group. Asterisks in FIG. 14 indicate significant differences from each control.

Other endpoint measurements were made. Ultimate infarct size may be affected by an increase or decrease in myocardial oxygen utilization. Two important determinants of myocardial oxygen compensation are heart rate and pressure load. Heart rate-pressure product (heart rate x mean arterial blood pressure) provides an approximation of the change in myocardial oxygen demand by the heart. Therefore, heart rate-pressure product was calculated to determine whether the observed decrease in infarct size was associated with a change in heart rate-pressure product. Heart rate and mean arterial pressure were continuously monitored throughout the experimental protocol and this data was used to calculate the heart rate-pressure product for the particular trial in the trial for each experimental group.

The risk area percentage of the left ventricle decreased in the ETC-216 treated heart as compared to the control for both acute and chronic treatment, but the results were not statistically significant. The percent infarct of the risk zone and the percent infarct of the left ventricle were significantly reduced in the ETC-216 treated heart compared to the control for both acute and chronic treatment. These results indicate that ETC-216 is cardioprotective when administered acutely and chronically.

Creatine kinase activity of myocardial tissue at risk and non-risk sites can be compared. The principle of the assay is based on the increase in absorbance of the reaction mixture at 340 nm, which is the result of equimolar reduction of NAD to NADH. Absorbance change rate is directly proportional to creatine kinase activity. One unit is defined as the amount of enzyme that produces one micromolar NADPH per minute under the conditions of the assay method.

Myocardial tissue applied to prolonged blood flow deficiency without reperfusion may undergo characteristic morphological changes of necrosis with the presence of inflammatory cells. The morphological appearance of ischemia-induced apoptosis is different from what appears as a result of reperfusion. The latter is characterized by a build band, called build band necrosis. Cardiac tissue from each group was preserved and prepared for examination by electron microscopy.

Ischemic reperfusion injury is associated with the accumulation of inflammatory cells, mainly neutrophils, in the danger zone. Myeloperoxidase (MPO) is an enzyme almost exclusively present in neutrophils (Liu et al., J. Pharmacol. Exp. Ther. 287: 527-537, 1998). Thus, it is expected that tissue from each site of the heart can be measured for MPO activity as an indicator of injury. It is also anticipated that interventions that can reduce the inflammatory response may be associated with a decrease in MPO activity at risk areas reperfused when compared to cardiac tissue from risk areas of non-treated animals. That is, the percentage change in risk (non-risk / non-risk site) in MPO activity can be reduced in the drug-treated heart as compared to the vehicle treated control heart.

At the end of the experiment, tissue myeloperoxidase (MPO) activity can be measured in a preliminary uncontrolled non-certified assay. Heart tissue samples were obtained from hazardous and non-hazardous sites, homogenized in 0.5% hexadecyltrimethyl ammonium bromide and dissolved in 50 mM potassium phosphate buffer (pH 6.0) (see also Liu et al., 1998, J). Pharmacol.Exp. Ther. 287: 527-537). Homogenates were centrifuged at 12.500 g at 4 ° C. for 30 minutes. The supernatant was collected and reacted with 0.167 mg / ml o-dianisidine dihydrochloride (Sigma) and 0.0005% H ₂ O _{2 in} 50 mM potassium phosphate buffer (pH 6.0). The change in absorbance was measured by spectrophotometry at 460 nm. One unit of MPO was defined as the amount of enzyme that hydrolyzes 1 mmol of H ₂ O ₂ per minute at 25 ° C. Although not presented herein, the results from this preliminary experiment appear to show no difference between ETC-216 and vehicle treated heart in terms of ischemic reperfusion injury, but this result is for example prior to ischemic reperfusion injury. Comparing MPO levels have not yet been certified.

ETC-216 treated heart was protected from ischemic reperfusion injury, as evidenced by the percent infarct of reduced risk area and the percent infarct of left ventricle. Cardioprotection is administered in two dosing protocols: ETC-216 administered at a single dose of 100 mg / kg before the onset of ischemia or ETC-216 administered at two doses of 100 mg / kg (one dose administered one day before ischemia, Dose was administered immediately before ischemia).

Example  5: LAD -Occluded Reperfusion  Determination of the Minimum Effective Amount for Acute Administration in Rabbit Hearts

This example demonstrates the prophylactic efficacy of various doses of ETC-216 when administered in a single pretreatment method immediately before the onset of focal ischemia. The test in Example 2 was focused on the effect of ETC-216 as a cardioprotectant in an in vivo test in which rabbit hearts were applied to focal myocardial ischemia for a period of 30 minutes followed by a minimum of 4 hours of reperfusion. Two dosage regimens were used. In the first protocol, ETC-216 was tested as a single pre-treatment with the circulatory system exposed to 100 mg / kg of drug immediately before the onset of focal ischemia, whereas in the second protocol (prior to and before the day 1). Two 100 mg / kg pretreatments were administered. Both regimens indicated that one or two treatments with 100 mg / kg ETC-216 were cardioprotective.

Thus, ETC-216 was tested with a single pretreatment method in which the heart was exposed to a single dose of medication or equivalent volume of vehicle immediately prior to the development of focal ischemia to determine the effect on cardioprotection. The heart was analyzed by the same method used in Example 2. The protocol is also designed to find the minimum effective amount of ETC-216 that treats the rabbit heart for protection from ischemia.

To find the minimum effective amount of ETC-216, the same protocol for acute treatment in which animals were single-treated with 10, 3 or 1 mg / kg ETC-216 or equivalent volume of vehicle, as shown in FIG. 16 (see FIG. 13) was used. For the 10 mg / kg treatment group, the risk area (AAR) or ischemic site expressed as a percentage of total left ventricle was similar in the control and treatment groups (FIG. 17). Rabbits treated with 10 mg / kg ETC-216 showed smaller infarcts (p <0.0005) expressed as a percentage of AAR compared to rabbits treated with vehicle (FIG. 18). A decrease in myocardial infarct size (p <0.0001) was also observed when the data were expressed as a percentage of total left ventricle (FIG. 17).

Similar results were observed with a dose of 3 mg / kg. AAR expressed as a percentage of total left ventricle was similar in the ETC-216-treated and vehicle-treated groups (FIG. 17). Rabbits treated with 3 mg / kg ETC-216 showed smaller infarcts (p <0.05) expressed as percentage of risk area compared to rabbits treated with vehicle (FIG. 17). A decrease in myocardial infarction size (p <0.05) was also observed when expressing data as a percentage of total left ventricle (FIG. 17).

Expressed as a percentage of the left ventricle, there was no significant difference in AAR size between ETC-216 and vehicle at the dose of 1 mg / kg (FIG. 17). At 1 mg / kg, no significant differences were seen between the groups in the percentage of AAR (FIG. 17), and the myocardial infarction size (FIG. 17) expressed in percent of total left ventricle.

A summary of the data obtained from each of the four acute treatment groups (ie 100, 10, 3 and 1 mg / kg) and their respective controls is shown in FIG. 17. Infarct AAR was similar in each of the four groups. Of the four dosing regimens, infarct size, expressed as a percentage of the risk site or as a percentage of the left ventricle, was reduced by ETC-216 doses of 100, 10 and 3 mg / kg compared to the respective controls. In contrast, the infarct size in the group of animals administered 1 mg / kg did not differ from that observed in each vehicle-treated group.

18 shows examples of transient changes in lipoprotein nonesterified cholesterol. Blood samples were obtained from rabbits immediately before and after administration of 1, 3, 10 or 100 mg / kg of ETC-216 or vehicle. Serum lipoproteins showed non-esterified cholesterol profiles obtained in representative transient serum samples, separated by size by gel-filtration chromatography with on-line non-esterified cholesterol assays. In spite of the substantial absence of non-esterified cholesterol in the ETC-216 test drug administered intravenously, 45 minutes after ETC-216 administration, an increase in high density cholesterol non-esterified cholesterol at 10 mg / kg or 10 At mg / kg there was a lesser increase. In addition, there was a marked marked delay in very low density lipoprotein non-esterified cholesterol 210 and 270 minutes after administration of 10 mg / kg or 100 mg / kg ETC-216. In addition, the change in lipoprotein non-esterified cholesterol was not evident with the 3 mg / kg ETC-216 treatment dose at the time point evaluated, but this dose was cardioprotective as shown in FIG. 17.

This result demonstrates that the 100 mg / kg, 10 mg / kg and 3 mg / kg doses are effective predictive doses of ETC-216.

Example  6: ETC -216 is occluded- Reperfusion  Rabbit in the heart LAD  Ischemia when administered after the manifestation of occlusion Reperfusion  Prevent damage.

This example demonstrates the efficacy of ETC-216 in preventing or reducing ischemic reperfusion injury when administered after an ischemic or obstructive attack. The test in Examples 2 and 3 is to illustrate the prophylactic effect of treating heart muscle prior to the onset of ischemia. Therefore, to determine whether ETC-216 can protect cardiac muscle after ischemia expression, LAD was occluded prior to administration of the test drug or vehicle. In this protocol, ETC-216 was tested in a single pretreatment method in which the heart was exposed to 10 mg / kg of drug or equivalent volume of vehicle administered during the last 5 minutes of focal ischemia and continuing over the first 55 minutes of reperfusion (FIG. 19). . The AAR or ischemic site expressed as a percentage of total left ventricle for the 10 mg / kg treatment group was similar in the control group (FIG. 20). Rabbits treated with ETC-216 showed a smaller (p <0.001) infarct expressed as a percentage of AAR compared to rabbits treated with vehicle (FIG. 20). A decrease in myocardial infarction size (p <0.0005) was also observed when expressing data as a percentage of total left ventricle (FIG. 20).

This example demonstrates that a single treatment administered after an ischemic attack alleviates or reduces ischemic reperfusion injury.

Various embodiments of the invention have been described. The description and examples are intended to illustrate the invention and are not limitative. Indeed, it will be apparent to those skilled in the art that modifications may be made to the various embodiments of the described invention without departing from the spirit of the invention or the scope of the appended claims below.

All documents cited herein are incorporated by reference in their entirety through their respective names.

Claims (74)

Apolipoprotein AI Milan: 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine complex (Apo A-IM: POPC complex) of about 1 mg to about 100 mg of protein per kg A method of treating acute coronary syndrome in a subject in need thereof, comprising administering at a dose. The method of claim 1, wherein the Apo A-IM: POPC complex is administered to the subject at a dose of about 15 mg / kg. The method of claim 1, wherein the Apo A-IM: POPC complex is administered to the subject at a dose of about 45 mg / kg. The method of claim 1, wherein the Apo A-IM: POPC complex is administered to the subject at a dose of about 15 mg / kg to about 45 mg / kg. The method of claim 1, wherein the Apo A-I Milan is a recombinant Apo A-I Milan. The apo A-IM: POPC complex according to claim 1, wherein the apo A-IM: POPC complex comprises apolipoprotein AI Milan and 1-palmitoyl-2-oleoyl- sn -glycero-3- at a ratio of about 1: 1 (protein wt / lipid wt). And consisting of phosphocholine. The method of claim 1, wherein the Apo A-IM: POPC complex is a sterile liquid pharmaceutical formulation. The method of claim 7, wherein the pharmaceutical formulation is administered to the subject at a dose of about 15 mg / kg. The method of claim 7, wherein the pharmaceutical formulation is administered to the subject at a dose of about 45 mg / kg. The method of claim 7, wherein the pharmaceutical formulation is administered to the subject once weekly for about 6 months, about 5 months, about 4 months, about 3 months, about 2 months, or about 1 month. The method of claim 7, wherein the pharmaceutical formulation is administered to the subject approximately daily for about one month. The method of claim 7, wherein the pharmaceutical formulation is administered to the subject approximately every 3 days for about 1 month to about 6 months. 8. The method of claim 7, wherein the pharmaceutical formulation is administered approximately every 10 days for about 1 month to about 6 months. 8. The method of claim 7, wherein the pharmaceutical formulation is administered approximately every 14 days for about 1 month to about 6 months. The method of claim 1, further comprising surgical intervention. The method of claim 15, wherein the surgical intervention comprises percutaneous coronary angioplasty or coronary artery bypass graft. The method of claim 1, further comprising administering another drug that treats, prevents or ameliorates a disease, disorder, symptom or pain associated with acute coronary syndromes. The method of claim 1, wherein the percent atheros volume in the diseased vessel of the subject is reduced by about −0.73% to about −1.29%. The method of claim 1, wherein the total atheroscopy volume in the subject's target vessel is reduced by about −15.1 mm 3 to about −12.6 mm 3. The method of claim 1, wherein the mean maximum atherosclerosis in the subject's target coronary artery section is reduced by about −0.039 mm to −0.044 mm. 8. The method of claim 7, wherein the pharmaceutical formulation is administered intravenously. The method of claim 21, wherein the pharmaceutical formulation is administered over about 1 hour. The method of claim 21, wherein the pharmaceutical formulation is administered over about 3 hours. 8. The method of claim 7, wherein the pharmaceutical formulation comprises Apo A-I Milano, POPC, sucrose-mannitol carrier and phosphate buffer. The method of claim 24, wherein the sucrose-mannitol carrier consists of about 6.0% to about 6.4% sucrose and about 0.8% to about 1% mannitol. The method of claim 25, wherein the sucrose-mannitol carrier consists of about 6.2% sucrose and about 0.9% mannitol. 8. The method of claim 7, wherein the pH of the pharmaceutical formulation is from about 7.0 to about 7.8. The method of claim 27, wherein the pH is about 7.5. 8. The method of claim 7, wherein the pharmaceutical formulation comprises about 12 mg / ml to about 18 mg / ml of Apo A-I Milano. 8. The method of claim 7, wherein the pharmaceutical formulation comprises about 11 mg / ml to about 17 mg / ml POPC. 8. The method of claim 7, wherein the pharmaceutical formulation comprises less than 6,000 particulates larger than 10 microns in size per 50 ml. 8. The method of claim 7, wherein the pharmaceutical formulation comprises less than 600 microparticles greater than 25 microns in size per 50 ml. 8. The method of claim 7, wherein the osmolality of the pharmaceutical formulation is from about 280 to about 320 mOsm. The method of claim 33, wherein the osmolality of the pharmaceutical formulation is about 290 mOsm. A method of reducing atherosclerosis plaque in a subject in need thereof comprising administering 45 mg / kg of the recombinant Apo A-I Milano: POPC complex to the subject at least once. 36. The method of claim 35, wherein the recombinant Apo A-I Milano: POPC complex is administered once a week, once a month or once a year. The method of claim 35, wherein the recombinant apo A-I Milano: POPC complex is administered once a week for about 3-5 weeks. The method of claim 35, further comprising administering a 15 mg / kg dose of the recombinant Apo A-I Milano: POPC complex to the subject at least once. The method of claim 38, wherein the complex is administered once a week, once a month or once a year. 36. The method of claim 35, wherein the subject is further treated with another drug that treats, prevents or ameliorates a disease, disorder, symptom or pain associated with acute coronary syndromes. The method of claim 40, wherein the drug is a statin, β-blocker, an ACE (angiotensin converting enzyme) inhibitor, an antithrombotic agent, an vasodilator, or a combination thereof. In a buffer comprising from about 6.0% to about 6.4% sucrose and further comprising from about 0.8 to about 1% mannitol and having a pH of about 7.0 to about 7.8, recombinant Apo AI Milan or variant / 1- Liquid pharmaceutical formulation comprising palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine complex. 43. The pharmaceutical formulation of claim 42, comprising about 6.2% sucrose. 43. The pharmaceutical formulation of claim 42, comprising about 0.9% of mannitol. 43. The pharmaceutical formulation of claim 42, wherein the pH is about 7.5. The pharmaceutical formulation of claim 42, wherein the concentration of recombinant Apo A-I Milano is from about 12 mg to about 18 mg per ml buffer. Recombinant Apo AI Milan or a variant thereof complexed with 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine and a pH drug comprising 2% glucose and 4 mM sodium phosphate A pharmaceutical formulation comprising 7.0 to about 7.8 phosphate buffers. 48. The pharmaceutical formulation of claim 47 comprising phosphate buffer to achieve a pH of about 7.5. 48. The pharmaceutical formulation of claim 42 or 47, wherein the recombinant Apo A-I Milan is a conservatively substituted Apo A-I Milan. 48. The pharmaceutical formulation of claim 42 or 47 wherein the recombinant Apo A-I Milano and POPC form a complex at a recombinant Apo A-I Milano: POPC (protein wt / lipid wt) ratio of 1: 0.95. 48. A pharmaceutical formulation according to claim 42 or 47 comprising less than 6,000 particulates larger than 10 microns in size per 50 ml. 48. A pharmaceutical formulation according to claim 42 or 47 comprising less than 600 particulates larger than 25 microns in size per 50 ml. 48. The pharmaceutical formulation of claim 42 or 47, wherein the osmolality is from about 280 to about 320 mOsm. The pharmaceutical formulation of claim 53 wherein the osmolality is about 290 mOsm. 48. A pharmaceutical formulation according to claim 42 or 47 which is sterile. 48. The pharmaceutical formulation of claim 42 or 47 which is frozen. 48. A pharmaceutical formulation according to claim 42 or 47 in a sterile vial, a sterile pre-filled bag or a pre-filled syringe. a) recombinant Apo AI Milan or variant thereof complexed with 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine; b) about 6.0% to about 6.4% sucrose; And c) about 0.8 to about 1% of mannitol Lyophilized pharmaceutical formulation comprising a. 59. The pharmaceutical formulation of claim 58, comprising about 6.2% sucrose. 59. The pharmaceutical formulation of claim 58, comprising about 0.9% of mannitol. The pharmaceutical formulation of claim 58 further comprising a buffer having a pH of about 7.5. 59. The pharmaceutical formulation of claim 58, wherein the recombinant Apo A-I Milan is a conservatively substituted Apo A-I Milan. 59. The pharmaceutical formulation of claim 58, wherein the concentration of POPC is about 11 mg / ml to about 17 mg / ml. 59. The pharmaceutical formulation of claim 58, wherein the recombinant Apo A-I Milano and POPC form a complex at a recombinant apo A-I Milano: POPC (protein wt / lipid wt) ratio of 1: 0.95. 59. The pharmaceutical formulation of claim 58, comprising less than 6,000 particulates larger than 10 microns in size per 50 ml. 59. The pharmaceutical formulation of claim 58, comprising less than 600 microparticles greater than 25 microns in size per 50 ml. 59. The pharmaceutical formulation of claim 58, wherein the osmolality is from about 280 to about 320 mOsm. The pharmaceutical formulation of claim 67 wherein the osmolality is about 290 mOsm. The pharmaceutical formulation of claim 58 which is sterile. 59. The pharmaceutical formulation of claim 58 in a sterile vial, a sterile pre-filled bag or a pre-filled syringe. Administering apolipoprotein AI Milan: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine complex (Apo A-IM: POPC complex) at a dose of about 15 mg protein per kg A method of treating ischemic reperfusion in a subject in need thereof. Administering apolipoprotein AI Milan: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine complex (Apo A-IM: POPC complex) at a dose of about 45 mg protein per kg A method of treating ischemic reperfusion in a subject in need thereof. 72. The method of claim 2, 8 or 71, further comprising administering 45 mg of protein per kg. 72. The method of claim 3, 9 or 72, further comprising administering 15 mg of protein per kg.

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